WO2012113948A1 - Method for determining genetic predisposition to parkinson's disease - Google Patents

Method for determining genetic predisposition to parkinson's disease Download PDF

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WO2012113948A1
WO2012113948A1 PCT/ES2012/000039 ES2012000039W WO2012113948A1 WO 2012113948 A1 WO2012113948 A1 WO 2012113948A1 ES 2012000039 W ES2012000039 W ES 2012000039W WO 2012113948 A1 WO2012113948 A1 WO 2012113948A1
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Agustín RUIZ LAZA
Mario EZQUERRA TRABALÓN
Antonio GONZÁLEZ PÉREZ
Javier GAYÁN GUARDIOLA
Francisco Jesús MORÓN CIVANTOS
José Miguel CARRASCO CALANCHA
Luis Miguel REAL NAVARRETE
Eduardo TOLOSA SARRÓ
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Fundación Alzheimur
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention is part of the procedures to aid in the diagnosis of complex diseases. Specifically, the present invention pertains to procedures based on genetic markers indicative of the predisposition of a subject to suffer from Parkinson's disease. More specifically, the procedure, in Vitro, focuses on the detection, individually or jointly, of SNPs in various genes for the prognosis of said predisposition, based on the genome of an individual.
  • Parkinson's disease is a neurodegenerative disorder of the central nervous system that is often disabling from a motor point of view, also affecting speech and other neurological functions.
  • PD affects 1% of the population at 50 years and 4% of the population at 85 years (1).
  • PD belongs to a group of disorders called “movement” and is characterized by the presence of muscle stiffness, tremor, slowness of movement (bradykinesia) and even complete loss of movement (aciniesia) in cases more extremes
  • the symptoms observed are results of a decrease in the stimulation of the motor cerebral cortex from the basal ganglia. Secondary symptoms may include cognitive disorders and language disorders.
  • the course of the disease is chronic and progressive.
  • PD phenocopies PD (individual or group of individuals in a population which lack a given genotype has the same phenotype (PD) than that which if it possesses said genotype) due to intoxications and infections of the central nervous system should not be ruled , the bulk of PD cases are considered "complex" forms of the disease whose etiology would be due to linear and non-linear integration processes of a multitude of genetic factors (low penetrance alleles) and non-genetic (environmental factors, basically toxic or Infectious agents).
  • SNCA alpha- synuclein, NM_000345) (3), the region of MAPT (microtubule- associated tau protein, NM_001123066) (4), NUCKS1 (nuclear casein kinase and cyclin -dependent, NM_022731) (5), PM20D1 (peptidase 20 domain containing 1 precursor, BC063477) (6), SLC41A1 (solute carrier family 41 member 1, N _173854) (7), BST1 (marrow stromal cell antigen 1 precursor, NM_004334 ) (8), LRRK2 (leucine-rich repeat kinase 2, NM_198578) (9), USP24 (ubiquitin specific protease 24, N _015306) (10), SLC6A3 (solute carrier family 6, NM_001044) (11) and GBA (glucocerebrosidase precursor, NM_000157) (12).
  • SNCA alpha- synuclein, NM
  • oligonucleotide that detect SNPs across the entire genome capable of identifying and genotyping of 10,000 or more SNPs in each individual, may reveal which SNPs are present or absent in patients and in controls of a specific genetic association study.
  • the data generated in these studies can be conceptualized as a table of values in which each column represents an individual genome, each line a certain SNP in said genomes, with a + or - symbol in each cell representing the presence or absence of the said SNP in each genome investigated.
  • HFCC computer application (WO2008010195) allows to determine genetic associations and identify genes that influence the appearance of any phenotypic trait, individually or jointly. This application allows you to filter, delete and select those associations that will be validated later in larger groups.
  • PINK1 PTEN induced putative kinase 1 precursor, NM_032409
  • HtrA2 HtrA serine peptidase 2 isoform 1 preproprotein, NM_013247
  • the present invention provides new methods for predicting, preclinically, the susceptibility to suffer PD of complex etiology, by employing new genetic markers (Single Nucleotide Polymorphisms, SNPs) or combinations thereof.
  • SNPs Single Nucleotide Polymorphisms
  • the present invention provides a new method of genetic scoring, based on the specific genotype of the loci of said genetic markers and others associated with the age of onset of the disease, which allows the classification of individuals in patients affected by complex PD No need for additional factors. Brief description of the figures.
  • Figure 1 Graphical representation by means of a Forest plot of the effect of the TT / CT diploma of the markers rs869026 and rsl0945364 in Parkinson's disease. Pooled: joint effect of the three series.
  • Y Relative Risk;
  • the inventors used the bioinformatic technology of HFCC meta-analysis (Hypothesis Free Clinical Cloning, WO2008010195) to select combinations of genetic markers distributed throughout the genome that are associated with Parkinson's disease.
  • stage I a complete genome scan was performed with HFCC applied to 272 cases and 272 controls.
  • the best couples made up of 384 independent markers were evaluated in the Spanish and North American population (Stage II and III). Only those couples who show a consistent effect on the three Studies undergo a meta-analysis (a study based on the structured and systematic integration of information obtained in different clinical studies, on a particular health problem, consisting of identifying and reviewing controlled studies on a certain problem, in order to give a synthetic quantitative estimate of all available studies.) (stage IV).
  • the term "genetic marker” and its plural refers to single nucleotide change polymorphisms (SNPs), from now on they will also be referred to as markers of the invention or SNPs of the invention interchangeably.
  • SNPs single nucleotide change polymorphisms
  • the present invention uses the meta-analysis of three independent series of cases of complex PD and unrelated controls. Two of the series are North American and were obtained from the public repositories of the National Center for Biotechnological Research (NCBI). The remaining series is Spanish and was obtained thanks to a collaboration with the Hospital Clinic of Barcelona. In total, the genotypes obtained from the DNA of 2697 independent individuals (1305 cases of complex PD and 1391 controls) were used for this study.
  • HFCC technology can be applied to select a set of diplomatic genetic markers (genotype combinations of two independent SNPs) to explain the differential risk that individuals have to suffer from PD.
  • markers although selected in pairs, retain the degree of association individually, so they can be used both individually, as in pairs, or together.
  • the public data of complete genomes can be used.
  • the repositories that guard said data will have been responsible for genotyping the samples of the individuals.
  • HFCC technology is applied through the use of solid devices or supports containing oligonucleotides capable of hybridizing with known SNPs. These devices are known in the art as SNPs or SNP Arrays chips and are commercially available. Using these starting materials, the genome of the selected individuals can be explored to identify all possible diplomotypic combinations. The appropriate filters are selected in the HFCC application.
  • HFCC systematically uses three filters in different phases of the study: a noise filter, which is a control-control comparison to eliminate spurious associations due to bad randomization of the diploma in the control group, an address filter that selects only those combinations in the that the direction of the effect is concordant and a filter tractor alleles (tracking ally filter) that identifies strongly deviated markers that introduce false positives.
  • the desired statistical parameters are included, for which the HFCC has a parallel computing algorithm. The selection of the desired markers is carried out automatically and subsequently, these SNPs are ordered and prioritized using either suitable software for this purpose or through conditional analysis strategies based on genes previously associated with PD. In this way, the best pairs of SNPs can be selected to design a validation test, which is carried out in series independent to purify the selection, eliminate false positives and retain the SNPs consistently associated with PD.
  • the gene sequences to be synthesized for the multiple genotyping assay are determined. These sequences must be suitable for simultaneous reading in the BeadXpress System.
  • the BeadXpress (I Ilumina inc, USA) technology consists of a 30nm resolution two-channel simultaneous non-confocal laser reader that allows you to read the holographic cylinders used in Veracode technology (I Ilumina inc, USA) (see below).
  • SNPs of the invention can be genotyped by an isolated sample of an individual, by contacting probes for SNPs with genomic DNA isolated from said sample, under appropriate conditions for hybridization of said probe to said gene.
  • Said probe can be a wild-type DNA (that is, the SNP under investigation is not present in the probe, and hybridization takes place when the SNP according to the invention is not found in the DNA in the sample), or a mutant (that is, transporting the SNP that is sought, and hybridization only takes place if the SNP is found in the DNA in the sample).
  • sample is understood as a portion of tissue or volume of body fluid, preferably peripheral blood, but which can also be any other tissue isolated from the body, for example by biopsy, or fluid sample body that contains the DNA of the individual under study.
  • tissue or volume of body fluid preferably peripheral blood
  • sample body that contains the DNA of the individual under study.
  • allelic probes to be used, their lengths, preferably between 50-300 bases, or the hybridization conditions. Sequences that flank the SNP can be used and that the expert will find in the sequence listing of the present invention.
  • Veracode is the technology of choice for the validation of HFCC results. This technology uses the power of digital holographic code to offer a robust detection system and is ideal for multiple tests that require high accuracy, reproducibility and speed.
  • the system uses microspheres differentiated by a holographic mark of high density (24-bit). When the VeraCode microspheres are excited by a laser, each one emits a unique image code, which allows a quick and specific detection by the reading system called "BeadXpress Reader System” (Illumina inc, USA). According to the level of multiplex required, the tests are created by mixing different microspheres (cylinders) with different codes, which can range from one to several hundred.
  • Each type of micro-sphere carries on the surface covalently bound oligonucleotide species specific to the region containing the polymorphism. Previously, said region is detected in each of the genomes to be studied using the Illumina GoldenGate system (Illumina Inc, USA). In this way, the system can simultaneously interrogate up to 384 single nucleotide polymorphisms (SNPs) per DNA sample.
  • the Goldengate system allows the genotyping of SNPs through a process of extension and ligation of "sequence-specific" DNA. The system allows to link only those oligos hybridized correctly. The correct fragments, once bound, are amplified with universal primers. At - * t - - - - - - -
  • a first object of the present invention is an in vitro method for determining the predisposition to suffer from Parkinson's disease in an isolated sample of an individual, comprising genotypic analysis of at least one SNP to be chosen from the group consisting of : rs577562, rs2986574, rsllll9097, rs4658673, rs4490160, rsl0496075, rsl2987286, rs847126, rs9036, rs6805546, rs4505714, rs7650598, rsl0155062, rs218280, rs869026, rs3094694, rsl549355, rs7747934, rs4583999, rsl0945
  • Table 2 shows the chromosomal location, the name of the gene and the access number of the SNPs of the invention while in Table 1 the relationship between the SNPs of the invention, the variation they present, and the sequence list are shown. of the invention, which include the flanking nucleotide sequences.
  • the in vitro method of the invention is useful for predicting the risk of complex PD from an isolated sample of an individual.
  • the in vitro method of the invention uses the SNPs rs869026 and rsl0945364, located on chromosomes 5 and 6 respectively.
  • SNPs rs869026 and rsl0945364 located on chromosomes 5 and 6 respectively.
  • Another aspect of the present invention provides an in vitro method for determining the predisposition to suffer from Parkinson's disease in an isolated sample of an individual, which comprises the genotypic analysis of at least one SNP to choose from the group consisting of: rsl039189, rsl2987286, rsl364658, rsl549355, rs218280, rs2332631, rs353111, rs394475169, rs3957751606 , rs761267, rs7650598, rs869026 and their combinations.
  • SNPs markers
  • a panel of markers SNPs
  • predictive capacity For the construction of said panel, a combination of SNPs associated with the "status" of cases is used (these are the markers present in Table 5) to which is also added a small subset of markers that present statistically significant indications of association with the age of onset of the disease ( ⁇ . ⁇ , age-at-onset) (Table 6).
  • Example 4 shows the creation of said predictive panel, which can be used as a genetic scoring system based on the genotypes of the loci, which allows the classification of affected individuals of complex PD without having to take into account any other factor.
  • of the present invention is an in vitro method based on a genetic scoring system comprising the joint genotypic analysis of the SNPs: rs577562, rs2986574, rsllll9097, rs4658673, rs4490160, rsl0496075, rsl2987126 , rs9036, rs6805546, rs4505714, rs7650598, rsl0155062, rs218280, rs869026, rs3094694, rsl549355, rs7747934, rs4583999, rsl0945364, rsll56143, rsl888349, rsl2353255, rs7027515, rs332185, rs763373, rsl0750861, rsll564173, rs2332631, rsl039189
  • a set of reagents or kit for the determination of the predisposition to suffer from parkinson's disease in an isolated sample of an individual, characterized is also the subject of the present invention.
  • the 541 available individuals were genotyped for 396,591 different SNPs distributed in the 22 autosomes. To do this they used two chips (SNP arrays Infinium I and II) of the Illumina company (Illumina Inc, USA). With this starting data, HFCC technology was used to explore all possible diplomotypic combinations of the 396,591 genotyped SNPs markers. As a result, an exhaustive screening of all existing civil patterns was carried out. In this way, the database available is divided into three replication groups of 90 cases and 90 controls (one of them had one more control) and the HFCC software was executed programming the statistical evaluation of all the diplomas (2-loci models M1, M2 and Mi 6 according to Li and Reich 2000 (7)).
  • the system selected 4.4% of the markers (17825 SNPs) that formed 26371 diploma combinations. These were ordered and prioritized using either Alambique software (21) or conditional analysis strategies (looking for pairs of markers that at least contained SNPs within genes previously associated with PD). The 384 most promising selected SNPs (table 2) were correspondingly validated in independent series (see below).
  • SNPs selected by HFCC Sense or forward chain data.
  • CV validation class
  • BV Validation tray
  • LGR Location Relating to Gen.
  • OPA Oligonucleotide Pooling Assay
  • the panel of markers selected during HFCC was sent to the company IIlumina Inc to perform a bioinformatic analysis and determine the gene sequences that will be synthesized for the multiple genotyping assay (OPA, oligonucleotide pooling assay).
  • Chr5_rs869026 x chr6_rsl0945364 as a genetic susceptibility factor for the disease after performing a complete bi-variant complete genome scan and two validation batches.
  • stage I the HFCC
  • two independent validations were used.
  • OR Odds ratio
  • This English term has been translated as ratio of possibilities, ratio of odds, odds ratio and is the ratio of two reasons: the numerator is the reason for the probability of an event happening and the probability that it does not happen under certain conditions and the denominator is the reason for the probability that such an event will happen and the probability that it will not happen under the complementary conditions.
  • the genomic marker rsl0945364 is found in a gene desert (that region of the genome where there are no known coding sequences) of the long arm of chromosome 6 (6q27). Therefore, no hypothesis can be established about the biological plausibility of this chromosomal region with respect to Parkinson's disease since it is unknown what functions this region contains.
  • the rs869026 marker located at 5q31.2, is in the 5 'region of the SILl gene. This gene is responsible for Marinesco-Sj ógren Syndrome and is a very strong candidate for complex neurodegenerative disorders. However, it is important to indicate that, to date, this gene had never been studied or associated for Parkinson's disease.
  • PLINK a tool set for whole-genome association and population-based linkage analyzes Purcell S, Neale B, Todd-Brown K. , Thomas L, Ferreira MA, Bender D, aller J, Sklar P, from Bakker PI, Daly MJ, Sham PC. Am J Hum Genet. 2007 Sep; 81 (3): 559-75).
  • the software allows the genetic evaluation of multiple markers and the performance of different genetic tests that serve as a measure of heterogeneity.
  • SNPs must maintain their association when we calculate their effect with a logistic regression adjusted for age and sex, ii) they must support an analysis stratified by study (Stages 1,2,3) iii) there must be absence of heterogeneity (Breslow index> 0.05) and iv) the meta-analysis adjusted for age and sex must be positive.
  • Table 5 shows the SNPs markers selected by HFCC and which have been validated by analyzing two additional independent series of cases and controls using different statistical methods.
  • the markers located on chromosome 12 rslll75655, rsll564173, rsll564203, rsll829088, rsl907632, rs2723264
  • This gene has been previously involved in PD (9).
  • its inclusion in the selection made by HFCC demonstrates the efficiency of the method applied.
  • the markers identified by conventional genotyping technologies such as PCR-RFLP, ARMS, real-time PCR or direct sequencing of PCR products etc. are genotyped.
  • ORconjunta average effect of each marker taking into account the three studies carried out.
  • Table 6 is the result of this second selection of markers and provides a total of 21 SNPs of the HFCC panel that are significantly associated (p ⁇ 0.05) to the age of PD onset (AAO).
  • This calculation was carried out with plink (PLINK: a tool set for whole-genome association and population-based linkage analyses. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, aller J, Sklar P, from Bakker PI, Daly MJ, Sham PC.Am J Hum Genet. 2007 Sep; 81 (3): 559-75) using linear regression (--linear) adjusting the effect by sex and by the series to which the patient.
  • SNP single nucleotide polymorphism.
  • CHR chromosome.
  • BP base pairs.
  • Al allele 1.
  • NMISS number of individuals available for the study.
  • BETA Beta linear regression coefficient.
  • Stat statistical value of the linear regression.
  • P significance value.
  • Table 8 shows the results of the discriminant analysis and cross-validation using the constructed function and issuing a score for each individual with the coefficients of Table 7.
  • the results demonstrate a predictive capacity of the control status of 72.8% ( cross validation 71.8%) and a predictive capacity of case status of 77.4% with a resulting cross validation of 75.7%. These estimates far exceed 50% (which is estimated to happen if it will be about predicting the condition of sick PD or healthy by throwing a coin in the air (that is by chance, table 8
  • the data obtained with the present invention indicate, therefore, that the combination of SNPs rs577562, rs2986574, rsllll9097, rs4658673, rs4490160, rsl0496075, rsl2987286, rs847126, rs9036, rs6805546, rs450575, rs5355995, rs650914, rs650914, rs6505145, rs4509145, rs4509145, rs4509146 , rs7747934, rs4583999, rsl0945364, rsll56143, rsl888349, rsl2353255, rs7027515, rs332185, rs763373, rs763373, rsl0750861, rsll564173, rs2332631, rsl0399 subset of individuals in the population at
  • the 532 controls of this validation were collected from pre-compiled panels in the Coriell repositories. All selected individuals expressed their status as Caucasian targets free of neuropsychiatric diseases and were questioned about neurological diseases such as Alzheimer's disease, amioatrophic lateral sclerosis, ataxias, autism, major depression, cerebrovascular diseases, dementia, Parkinson's disease and schizophrenia . His parents and siblings did not have neurological diseases either. The average age of these controls is 58 years (15-98 years).
  • Genotypes belonging to these individuals were extracted directly from the matrices obtained from NCBI using conventional computer technologies. Specifically, we obtained the 384 markers mentioned in table 2 by successively applying to the NCBI matrices the commands -proxy-impute all (to infer the markers that were not present), --snp (to select from the matrix the list of SNPs from interest) and -make-bed (to generate a new data matrix exclusively with the selected markers).
  • the raw genotypes obtained from the matrices were used as input for the HFCC software and other bioinformatics programs, such as PLINK, which are used to evaluate the results of the study.
  • PLINK (6) and in particular the -assoc command, allows the univariate study of each SNP with respect to the disease.
  • the -linear command allows the adjusted linear regression analysis.
  • the -logistic command allows the logistic regression analysis adjusted by age and sex, the - within and -homog commands allow the calculation of the heterogeneity index and the Breslow-Day statistic and the stratified analysis by series.
  • the --meta-analysis command allows the joint analysis of all studies.
  • HFCC is used for bi-variant analysis.
  • the plot of Forest is done with the free episheet software
  • Clark IE Dodson MW, Jiang C, Cao JH, Huh JR, Seol JH, et al. Drosophila pinkl is required for mitochondrial function and interacts genetically with parkin. Nature 2006 4 1 (7097): 1162-6.
  • BAP a mammalian BiP- associated protein, is a nucleotide exchange factor that regulates the ATPase activity of BiP. J Biol Chem. 2002; 277 (49): 7557-63.

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Abstract

The present invention provides a method for predicting an individual's predisposition to suffering from Parkinson's disease (PD) on the basis of a blood sample isolated from said individual. Said method comprises combined use of 36 novel SNPs with the ability to pre-clinically predict the appearance of PD complex. Furthermore, the present invention provides a method that comprises the use of a digenic combination of SNPs from the SIL1 × Chr6 loci, which confers on carriers thereof a greater risk of suffering from Parkinson's disease.

Description

PROCEDIMIENTO PARA LA DETERMINACIÓN DE LA PREDISPOSICIÓN GENÉTICA A LA ENFERMEDAD DE PARKINSON PROCEDURE FOR THE DETERMINATION OF GENETIC PREDISPOSITION TO PARKINSON'S DISEASE
Campo técnico  Technical field
La presente invención se enmarca dentro de los procedimientos para ayuda al diagnóstico de enfermedades complejas. En concreto, la presente invención pertenece la los procedimientos basados en marcadores genéticos indicativos de la predisposición de un sujeto a sufrir la enfermedad de Parkinson. Más concretamente, el procedimiento, in Vitro, se centra en la detección, individual o conjunta, de SNPs en diversos genes para el pronóstico de dicha predisposición, en base al genoma de un individuo. The present invention is part of the procedures to aid in the diagnosis of complex diseases. Specifically, the present invention pertains to procedures based on genetic markers indicative of the predisposition of a subject to suffer from Parkinson's disease. More specifically, the procedure, in Vitro, focuses on the detection, individually or jointly, of SNPs in various genes for the prognosis of said predisposition, based on the genome of an individual.
Estado de la técnica State of the art
La enfermedad de Parkinson (PD) es un desorden neurodegenerativo del sistema nervioso central que a menudo es incapacitante desde un punto de vista motriz afectando, también, al habla y otras funciones neurológicas .  Parkinson's disease (PD) is a neurodegenerative disorder of the central nervous system that is often disabling from a motor point of view, also affecting speech and other neurological functions.
Desde un punto de vista epidemiológico PD afecta al 1% de la población a los 50 años y a un 4% de la población a los 85 años ( 1 ) .  From an epidemiological point of view PD affects 1% of the population at 50 years and 4% of the population at 85 years (1).
Desde un punto de vista clínico, PD pertenece a un grupo de trastornos denominado "del movimiento" y se caracteriza por la presencia de rigidez muscular, temblores, lentitud de movimientos (bradicinesia) e incluso la pérdida completa de movimientos (aciniesia) en los casos más extremos. Los síntomas observados son resultados de una disminución de la estimulación de la corteza cerebral motora proveniente de los ganglios básales. Los síntomas secundarios pueden incluir alteraciones cognitivas y trastornos del lenguaje. El curso de la enfermedad es crónico y progresivo. Desde un punto de vista patogénico observamos un defecto de estimulación de la corteza cerebral que es debido a una deficiencia funcional y/o anatómica (pérdida física de neuronas dopaminérgicas ) en la sustancia negra que genera, en consecuencia, un déficit de la acción de la dopamina que es el principal neurotransmisor producido en dicha región cerebral . From a clinical point of view, PD belongs to a group of disorders called "movement" and is characterized by the presence of muscle stiffness, tremor, slowness of movement (bradykinesia) and even complete loss of movement (aciniesia) in cases more extremes The symptoms observed are results of a decrease in the stimulation of the motor cerebral cortex from the basal ganglia. Secondary symptoms may include cognitive disorders and language disorders. The course of the disease is chronic and progressive. From a pathogenic point of view we observe a defect in the stimulation of the cerebral cortex that is due to a functional and / or anatomical deficiency (physical loss of dopaminergic neurons) in the black substance that generates, consequently, a deficit in the action of the dopamine that is the main neurotransmitter produced in said brain region.
Al contrario que ocurre en la patogénesis de PD, las bases etiológicas de la enfermedad no están bien establecidas. La mayoría de los casos de PD se consideran idiopáticos (de causa desconocida) . No obstante, hay descritas una minoría de formas familiares de la patología que son debidas a defectos discretos (mutaciones) en genes autosómicos ya identificados como PARK2 (parkin, NM_004562) , SNCA (alfa-sinucleína, NM_000345 ) , LRRK2 ( leucine-rich repeat kinase 2, NM_198578), UCHL1 (ubiquitin carboxyl-terminal esterase Ll, NM_004181 ) , GBA (glucocerebrosidase precursor, NM_000157) , FGF20, (fibroblast growth factor 20, NM_019851), ó MAPT (microtubule-associated protein tau (NM_001123066) entre otros (para una revisión detallada de los genes mendelianos véase OMIM número 168600 Unlike the pathogenesis of PD, the etiological basis of the disease is not well established. Most cases of PD are considered idiopathic (of unknown cause). However, a minority of familiar forms of the pathology are described that are due to discrete defects (mutations) in autosomal genes already identified as PARK2 (parkin, NM_004562), SNCA (alpha-synuclein, NM_000345), LRRK2 (leucine-rich repeat kinase 2, NM_198578), UCHL1 (ubiquitin carboxyl-terminal esterase Ll, NM_004181), GBA (glucocerebrosidase precursor, NM_000157), FGF20, (fibroblast growth factor 20, NM_019851), or MAPT (microtubule-associated protein ta30 (NM) among others NM306) (for a detailed review of Mendelian genes see OMIM number 168600
(http://www.ncbi.nlm.nih.gov/omim/168600; consulta realizada el 10 de enro de 2011). Aunque la existencia de fenocopias de PD (individuo o grupo de individuos de una población que, careciendo de un genotipo dado posee el mismo fenotipo (PD) que aquél que si posee dicho genotipo) debidas a intoxicaciones e infecciones del sistema nervioso central tampoco debe descartarse, el grueso de los casos de PD se consideran formas "complejas" de la enfermedad cuya etiología sería debida a procesos de integración lineales y no lineales de multitud de factores genéticos (alelos de baja penetrancia) y no genéticos (factores ambientales, básicamente tóxicos o agentes infecciosos). En otras enfermedades neurodegenerativas, como el síndrome de Marinesco-Sj órgren (MSS) , se ha demostrado que las mutaciones del gen sill (homólogo de SILl protein precursor, NM_022464) causan el fenotipo neurodegenerativo woozy (wz) de ratón (34) . Este hallazgo es importante pues estableció un vinculo directo entre una disfunción del retículo endoplásmico (ER) y la neurodegeneración (32, 33) . Se sospecha que el estrés del ER podría ser una de las causas principales de las enfermedades neurodegenerativas comunes (Enfermedad de Alzheimer y Enfermedad de Parkinson) pero hasta la fecha ninguna variante genética del gen SILl ha sido relacionada con el riesgo de ninguna de estas enfermedades. (http://www.ncbi.nlm.nih.gov/omim/168600; consultation made on January 10, 2011). Although the existence of phenocopies PD (individual or group of individuals in a population which lack a given genotype has the same phenotype (PD) than that which if it possesses said genotype) due to intoxications and infections of the central nervous system should not be ruled , the bulk of PD cases are considered "complex" forms of the disease whose etiology would be due to linear and non-linear integration processes of a multitude of genetic factors (low penetrance alleles) and non-genetic (environmental factors, basically toxic or Infectious agents). In other neurodegenerative diseases like syndrome Marinesco-Sj Orgren (MSS), it has been shown that mutations in the sill gene (homologous Sill protein precursor, NM_022464) cause the neurodegenerative phenotype woozy (wz) mouse (34). This finding is important because it established a direct link between endoplasmic reticulum dysfunction (ER) and neurodegeneration (32, 33). It is suspected that ER stress could be one of the main causes of common neurodegenerative diseases (Alzheimer's disease and Parkinson's disease) but to date no genetic variant of the SILl gene has been related to the risk of any of these diseases.
Evidentemente, el conocimiento actual de los factores genéticos y no genéticos que determinan la susceptibilidad a padecer PD es muy escaso, lo que dificulta el diagnóstico temprano de la patología, la identificación de las poblaciones en riesgo de padecer PD y el desarrollo de más y mejores métodos de diagnóstico y terapias.  Obviously, current knowledge of the genetic and non-genetic factors that determine susceptibility to PD is very scarce, which makes it difficult to diagnose diseases early, identify populations at risk of PD and develop more and better Diagnostic methods and therapies.
Para solucionar este difícil problema, la comunidad científica ha invertido considerables esfuerzos en las siguientes estrategias:  To solve this difficult problem, the scientific community has invested considerable efforts in the following strategies:
a. Genética aplicada a PD compleja  to. Genetics applied to complex PD
Existen numerosos trabajos que emplearon las técnicas genéticas para tratar de elucidar las bases etiológicas de PD compleja. Las enfermedades complejas son poligénicas o multifactoriales o el resultado final de efectos complejos de varios (o muchos) genes interaccionando con los factores ambientales. Para efectuar un rastreo sistemático de los estudios genéticos de PD compleja contamos con una base de datos (PDgene datábase, http://www.pdgene.org/; fecha de consulta 11 de Enero de 2011) que recoge la mayoría de los estudios de asociación genética. Actualmente, se contabilizan 764 estudios independientes que han analizado 2407 marcadores genéticos en 547 genes independientes con resultados muy dispares. En la actualidad los diez genes más consistentemente asociados a la PD compleja son SNCA (alpha- synuclein, NM_000345) (3), la región de MAPT (microtubule- associated protein tau, NM_001123066 ) ( 4 ) , NUCKS1 (nuclear casein kinase and cyclin-dependent , NM_022731) (5), PM20D1 (peptidase 20 domain containing 1 precursor, BC063477) (6) , SLC41A1 (solute carrier family 41 member 1, N _173854) (7) , BST1 (marrow stromal cell antigen 1 precursor, NM_004334) (8), LRRK2 (leucine-rich repeat kinase 2, NM_198578) (9) , USP24 (ubiquitin specific protease 24, N _015306) (10) , SLC6A3 (solute carrier family 6, NM_001044) (11) y GBA (glucocerebrosidase precursor, NM_000157) (12). Es interesante destacar que si bien la mayoría de los estudios realizados hasta la fecha han utilizado como estrategia el análisis de genes candidatos (candidate gene approach) , los resultados más consistentes han sido obtenidos utilizando técnicas de rastreo completo del genoma (GWAS, genomewide association studies). No obstante, la inconsistencia y variabilidad de resultados demuestra la dificultad que supone tratar de elucidar las bases genéticas de PD compleja (forma más común de PD y que es debida a la conjunción de varios genes y factores ambientales) . Es importante aclarar que la base de datos PDgene sólo recoge estudios univariantes (de un solo marcador genético) y no contiene resultados de estudios de interacción gen-gen (epistasis) , de interacción gen-ambiente o modelos causales multivariantes de la enfermedad. There are numerous works that used genetic techniques to try to elucidate the etiological bases of complex PD. Complex diseases are polygenic or multifactorial or the end result of complex effects of several (or many) genes interacting with environmental factors. To systematically track the genetic studies of complex PD we have a database (PDgene datábase, http://www.pdgene.org/; consultation date January 11, 2011) that includes most of the studies of genetic association Currently, there are 764 independent studies that have analyzed 2407 markers. genetic in 547 independent genes with very different results. At present the ten genes most consistently associated with PD are complex SNCA (alpha- synuclein, NM_000345) (3), the region of MAPT (microtubule- associated tau protein, NM_001123066) (4), NUCKS1 (nuclear casein kinase and cyclin -dependent, NM_022731) (5), PM20D1 (peptidase 20 domain containing 1 precursor, BC063477) (6), SLC41A1 (solute carrier family 41 member 1, N _173854) (7), BST1 (marrow stromal cell antigen 1 precursor, NM_004334 ) (8), LRRK2 (leucine-rich repeat kinase 2, NM_198578) (9), USP24 (ubiquitin specific protease 24, N _015306) (10), SLC6A3 (solute carrier family 6, NM_001044) (11) and GBA (glucocerebrosidase precursor, NM_000157) (12). It is interesting to note that although most studies conducted to date have used the candidate gene approach as a strategy, the most consistent results have been obtained using full genome tracking techniques (GWAS). ). However, the inconsistency and variability of results demonstrates the difficulty of trying to elucidate the genetic basis of complex PD (most common form of PD and that is due to the conjunction of several genes and environmental factors). It is important to clarify that the PDgene database only collects univariate studies (of a single genetic marker) and does not contain results of studies of gene-gene interaction (epistasis), of gene-environment interaction or multivariate causal models of the disease.
b. Análisis multilocus en la enfermedad de Parkinson. La integración de múltiples elementos causales en un modelo multivariante (multilocus en el caso de tratarse de genes) es una asignatura pendiente de la genética molecular humana en la actualidad. Existen intentos tempranos de la aplicación de modelos multivariantes a la enfermedad de Parkinson. En concreto, Lesnick y colaboradores (2007) (13) utilizaron una combinación del rastreo completo del genoma (GWAS) junto con un abordaje de rutas bioquímicas para tratar de demostrar que la maquinaria bioquímica encargada del enrutamiento de axones neuronales estaba involucrada en la etiología de PD. Sin embargo el modelo desarrollado no superó tan siquiera la primera replicación independiente (es decir que no se pudieron reproducir los resultados obtenidos cuando se aplicaron los mismos métodos y marcadores genéticos a otra población diferente) (14) . Además, los datos genéticos de PD compleja que se han hecho públicos y están disponibles para investigación se han utilizado para explorar el fenómeno de la epistasis y desarrollar nuevas técnicas de análisis mult ivariante (1, 15-19) . Los estudios mencionados son exploratorios y no incluyen las series de replicación que son fundamentales para la confirmación de los hallazgos obtenidos . b. Multilocus analysis in Parkinson's disease. The integration of multiple causal elements in a multivariate model (multilocus in the case of genes) is a pending subject of human molecular genetics today. There are early attempts at application of multivariate models to Parkinson's disease. Specifically, Lesnick et al (2007) (13) used a combination of full crawl genome (GWAS) with an approach of biochemical pathways to try to demonstrate that the biochemical machinery responsible for the routing of neuronal axons was involved in the etiology of P.S. However, the developed model did not even exceed the first independent replication (that is, the results obtained when the same methods and genetic markers were applied to a different population could not be reproduced) (14). In addition, the genetic data of complex PD that have been made public and available for research have been used to explore the phenomenon of epistasis and develop new multivariate analysis techniques (1, 15-19). The studies mentioned are exploratory and do not include the replication series that are fundamental for the confirmation of the findings obtained.
La idea básica subyacente a todos los estudios de asociación genética es que tanto las características fenotípicas normales, como los rasgos clínicos de las enfermedades, son debidos a una interacción o combinación de factores ambientales que operan sobre un fondo genético particular e individual.  The basic idea behind all genetic association studies is that both normal phenotypic characteristics, and clinical features of diseases, are due to an interaction or combination of environmental factors that operate on a particular and individual genetic background.
Actualmente, se considera que la mejor estrategia para los estudios de asociación genética es la identificación de genes empleando el rastreo completo del genoma ("shot gun approach", junto con estudios de asociación en el genoma completo) . En esta estrategia, que depende del desequilibrio de unión (Cuando un marcador genético está muy cerca, dentro de la secuencia de un cromosoma, de la mutación causante de una enfermedad, habitualmente se heredan conjuntamente. Este fenómeno es conocido como desequilibrio de unión, LD) , se emplean múltiples marcadores a lo largo del genoma comparando individuos no relacionados pero que presentan el rasgo en estudio, contra controles que no presentan dicho rasgo. En la actualidad es posible el examen del genoma completo de un individuo empleando productos comerciales de investigación genética basados en microdispositivos de oligonucleotidos que detectan SNPs a lo largo de todo el genoma con capacidad de identificación y genotipado de 10.000 o más SNPs en cada individuo, pudiendo revelar qué SNPs están presentes o ausentes en enfermos y en controles de un determinado estudio de asociación genética. Los datos generados en estos estudios pueden ser conceptualizados como una tabla de valores en la que cada columna representa un genoma individual, cada linea un determinado SNP en dichos genomas, con un símbolo + ó - en cada celda representando la presencia o ausencia del citado SNP en cada genoma investigado. Múltiples programas informáticos como por ejemplo Sumstat (Ott J, Hoh J. , 2003), o PLink (Purcell et al., American Journal of Human Genetics, 81) pueden analizar estos datos con objeto de aislar loci correlacionados estadísticamente con el rasgo en estudio. Estos mismos programas, además, pueden calcular la probabilidad de que dicha asociación sea real o un artefacto de los datos. Finalmente, recurriendo al mapa del genoma humano es posible delinear una hipótesis más refinada basada en la información sobre los genes cercanos al marcador asociado y diseñar estrategias de confirmación o validación de los resultados obtenidos. Este abordaje se ha utilizado en el estudio de rasgos monogénicos. Pero también es posible aplicarlo a rasgos complejos (poligénicos ) incluyendo aquellos en los que un gen individual tiene sólo un efecto muy pequeño e incluso indetectable por sí solo. Véanse como ejemplos: Hoh et al, 2003; o Marchini et al, 2005. La aplicación informática HFCC (WO2008010195) permite determinar asociaciones genéticas e identificar genes que influyen en la aparición de un rasgo fenotipico cualquiera, de forma individual o conjunta. Esta aplicación permite filtrar, eliminar y seleccionar aquellas asociaciones que se validarán posteriormente en grupos de mayor tamaño. Currently, the best strategy for genetic association studies is considered to be the identification of genes using complete genome tracking ("shot gun approach", along with association studies in the entire genome). In this strategy, which depends on the union imbalance (When a genetic marker is very close, within the sequence of a chromosome, the mutation causing a disease, they are usually inherited together. This phenomenon is known as union imbalance, LD ) , be they use multiple markers throughout the genome comparing unrelated individuals but who present the trait under study, against controls that do not have said trait. Currently examining the entire genome of an individual is possible using commercial products from genetic research based microdevices oligonucleotide that detect SNPs across the entire genome capable of identifying and genotyping of 10,000 or more SNPs in each individual, may reveal which SNPs are present or absent in patients and in controls of a specific genetic association study. The data generated in these studies can be conceptualized as a table of values in which each column represents an individual genome, each line a certain SNP in said genomes, with a + or - symbol in each cell representing the presence or absence of the said SNP in each genome investigated. Multiple computer programs such as Sumstat (Ott J, Hoh J., 2003), or PLink (Purcell et al., American Journal of Human Genetics, 81) can analyze this data in order to isolate loci statistically correlated with the trait being studied. . These same programs, in addition, can calculate the probability that said association is real or an artifact of the data. Finally, using the human genome map it is possible to delineate a more refined hypothesis based on the information about the genes close to the associated marker and design confirmation or validation strategies for the results obtained. This approach has been used in the study of monogenic traits. But it is also possible to apply it to complex (polygenic) features including those in which an individual gene has only a very small and even undetectable effect on its own. See as examples: Hoh et al, 2003; or Marchini et al, 2005. The HFCC computer application (WO2008010195) allows to determine genetic associations and identify genes that influence the appearance of any phenotypic trait, individually or jointly. This application allows you to filter, delete and select those associations that will be validated later in larger groups.
Al margen de los problemas relacionados con la exploración de las interacciones empleando datos de genoma completo, entre los que destacan la multidimensionalidad del análisis (el alto número de combinaciones a explorar conlleva un alto riesgo de falsos positivos por azar derivados de las múltiples combinaciones analizadas), la ausencia de consenso en cuanto a los modelos de interacción génica susceptibles de ser explorados y los problemas de interpretación de los resultados pues nuestro conocimiento sobre las relaciones moleculares es parcial, existen un buen número de ejemplos de trabajos que tratan de explorar la epistasis (efecto debido a interacción no lineal de dos mutaciones) entre dos genes y su relación con PD compleja. La mayoría de estos estudios tratan de establecer una conexión molecular o estadística entre dos genes independientes y la enfermedad en cuestión. Como en el caso anterior, los estudios suelen carecer de las series de replicación y sin ellas, el estudio no puede considerarse concluyente. Respecto a las interacciones moleculares, tal vez los trabajos más destacados en este campo sean los que establecieron la relación molecular entre dos genes asociados a PD familiar (20-23) y el establecimiento de una ruta interactiva de elementos génicos relacionados con el control de la toxicidad del manganeso y que incluía los genes SNCA (alpha-synuclein, NM_000345) y PARK9 (ATPase type 13A2 isoform, NM_022089) , ambos previamente asociados a PD (24) . También merecen destacarse los estudios moleculares que confirmaron que PINK1 (PTEN induced putative kinase 1 precursor, NM_032409) era epistático respecto de HtrA2 (HtrA serine peptidase 2 isoform 1 preproprotein, NM_013247) (25) . Apart from the problems related to the exploration of the interactions using complete genome data, among which the multidimensionality of the analysis stands out (the high number of combinations to explore entails a high risk of false positives by chance derived from the multiple combinations analyzed) , the absence of consensus as to the models of gene interaction that can be explored and the problems of interpretation of the results because our knowledge about molecular relationships is partial, there are a good number of examples of works that try to explore epistasis ( effect due to non-linear interaction of two mutations) between two genes and their relationship with complex PD. Most of these studies try to establish a molecular or statistical connection between two independent genes and the disease in question. As in the previous case, studies usually lack replication series and without them, the study cannot be considered conclusive. Regarding molecular interactions, perhaps the most outstanding works in this field are those that established the molecular relationship between two genes associated with familial PD (20-23) and the establishment of an interactive path of gene elements related to the control of manganese toxicity and that included the genes SNCA (alpha-synuclein, NM_000345) and PARK9 (ATPase type 13A2 isoform, NM_022089), both previously associated with PD (24). Also worth mentioning are molecular studies that confirmed that PINK1 (PTEN induced putative kinase 1 precursor, NM_032409) was epistatic with respect to HtrA2 (HtrA serine peptidase 2 isoform 1 preproprotein, NM_013247) (25).
Respecto a las interacciones gen-gen estadísticas destacamos los estudios de interacción digénica entre variaciones raras de los genes PRKN y LRRK2 (26) . También se ha publicado estudios de interacción de variaciones comunes para PD compleja. Por ejemplo, se ha descrito que los genes FGF20 y MAOB interaccionan para provocar PD (27). Igualmente se ha descrito que USP24 puede actuar sinérgicamente con USP40 ó UCHLl (28). Hay también descripciones de combinaciones genotípicas de SNCA y MAPT que incrementan el riesgo de padecer PD (29) . Otros autores han observado la interacción de IL-6 y ESR2 a nivel genético (30). Finalmente, se ha sugerido que la combinación de variantes genéticas en loci de detoxificación hepática como CYP2D6 y GSTM1 podría conferir un riesgo diferencial de padecer PD (31). Es prudente comentar nuevamente que ninguno de los estudios mencionados incluye replicaciones en series independientes.  Regarding the gene-gene statistical interactions, we highlight the studies of digénic interaction between rare variations of the PRKN and LRRK2 genes (26). Studies of interaction of common variations for complex PD have also been published. For example, it has been described that the FGF20 and MAOB genes interact to cause PD (27). It has also been described that USP24 can act synergistically with USP40 or UCHLl (28). There are also descriptions of genotypic combinations of SNCA and MAPT that increase the risk of PD (29). Other authors have observed the interaction of IL-6 and ESR2 at the genetic level (30). Finally, it has been suggested that the combination of genetic variants in liver detoxification loci such as CYP2D6 and GSTM1 could confer a differential risk of PD (31). It is prudent to comment again that none of the studies mentioned include replications in independent series.
Según todo lo anteriormente expuesto, no existen actualmente marcadores para la PD compleja, que puedan predecir anticipadamente la susceptibilidad a padecer la enfermedad. La presente invención proporciona nuevos métodos para predecir, preclínicamente, la susceptibilidad a padecer PD de etiología compleja, mediante el empleo de nuevos marcadores genéticos (Polimorfismos de un único Nucleótido, SNPs) o combinaciones de estos. Además, la presente invención proporciona un nuevo método de puntuación genética, basado en el genotipo específico de los loci de dichos marcadores genéticos y de otros asociados a la edad de comienzo de la enfermedad, que permite la clasificación de individuos en pacientes afectados de PD compleja sin necesidad de factores adicionales . Breve descripción de las figuras . According to all of the above, there are currently no markers for complex PD, which can predict in advance the susceptibility to the disease. The present invention provides new methods for predicting, preclinically, the susceptibility to suffer PD of complex etiology, by employing new genetic markers (Single Nucleotide Polymorphisms, SNPs) or combinations thereof. In addition, the present invention provides a new method of genetic scoring, based on the specific genotype of the loci of said genetic markers and others associated with the age of onset of the disease, which allows the classification of individuals in patients affected by complex PD No need for additional factors. Brief description of the figures.
Figura 1 : Representación gráfica mediante un plot de Forest del efecto del diplotipo TT/CT de los marcadores rs869026 y rsl0945364 en la Enfermedad de Parkinson. Pooled: efecto conjunto de las tres series. Y: Riesgo Relativo; X Meta- análisis. OR=1,780 [1,081-2,930], p=0,036. Análisis Conjunto: 0R=1, 66 [1, 36-2, 01] , p=3,66xE7  Figure 1: Graphical representation by means of a Forest plot of the effect of the TT / CT diploma of the markers rs869026 and rsl0945364 in Parkinson's disease. Pooled: joint effect of the three series. Y: Relative Risk; X Meta-analysis. OR = 1,780 [1,081-2,930], p = 0.036. Joint Analysis: 0R = 1.66 [1, 36-2, 01], p = 3.66xE7
Figura 2 : Curva COR para el análisis conjunto. Linea recta continua: linea de referencia; Linea curva punteada: variables; Linea curva discontinua: SNPs; Linea curva continua: series de sexo y edad. Y: Sensibilidad; X: Especificidad (por 0=0%, 1=100%, 0.5=50%). Los segmentos diagonales son producidos por los empates (un empate en el análisis discriminante acontece cuando un miembro del grupo de casos tiene la misma puntuación que otro del grupo de controles ) . Figure 2: COR curve for the joint analysis. Continuous straight line: reference line; Dotted curve line: variables; Dashed curve line: SNPs; Continuous curve line: sex and age series. Y: Sensitivity; X: Specificity (by 0 = 0%, 1 = 100%, 0.5 = 50%). Diagonal segments are produced by ties (a tie in the discriminant analysis occurs when a member of the case group has the same score as another member of the control group).
Descripción detallada de la invención Detailed description of the invention
Los inventores emplearon la tecnología bioinformática de meta-análisis HFCC (Hypothesis Free Clinical Cloning, WO2008010195) para seleccionar combinaciones de marcadores genéticos distribuidos por todo el genoma que se asocian con la enfermedad de Parkinson. The inventors used the bioinformatic technology of HFCC meta-analysis (Hypothesis Free Clinical Cloning, WO2008010195) to select combinations of genetic markers distributed throughout the genome that are associated with Parkinson's disease.
El esquema general para la identificación de parejas de marcadores asociadas a la enfermedad de Parkinson, ha sido el siguiente: Durante el primer paso (Stage I) se realizó un rastreo completo del genoma con HFCC aplicado a 272 casos y 272 controles. Las parejas mejores conformadas por 384 marcadores independientes se evalúaron en la población Española y Norte-americana (Stage II y III) . Sólo aquellas parejas que muestran un efecto consistente en los tres estudios se someten a un metaanálisis (un estudio basado en la integración estructurada y sistemática de la información obtenida en diferentes estudios clínicos, sobre un problema de salud determinado, consistente en identificar y revisar los estudios controlados sobre un determinado problema, con el fin de dar una estimación cuantitativa sintética de todos los estudios disponibles.) (stage IV). The general scheme for the identification of pairs of markers associated with Parkinson's disease has been as follows: During the first step (Stage I) a complete genome scan was performed with HFCC applied to 272 cases and 272 controls. The best couples made up of 384 independent markers were evaluated in the Spanish and North American population (Stage II and III). Only those couples who show a consistent effect on the three Studies undergo a meta-analysis (a study based on the structured and systematic integration of information obtained in different clinical studies, on a particular health problem, consisting of identifying and reviewing controlled studies on a certain problem, in order to give a synthetic quantitative estimate of all available studies.) (stage IV).
A efectos de la presente invención, la expresión "marcador genético" y su plural, se refiere a polimorfismos de un cambio de nucleótido único (SNPs), a partir de ahora se hará referencia a ellos también como marcadores de la invención o SNPs de la invención indistintamente. El análisis individual o combinado de los SNPs de la invención no había sido vinculado anteriormente con la susceptibilidad a padecer PD.  For the purposes of the present invention, the term "genetic marker" and its plural refers to single nucleotide change polymorphisms (SNPs), from now on they will also be referred to as markers of the invention or SNPs of the invention interchangeably. The individual or combined analysis of the SNPs of the invention had not previously been linked to susceptibility to PD.
A diferencia de estudios anteriores, la presente invención utiliza el meta-análisis de tres series independientes de casos de PD compleja y controles no relacionados. Dos de las series son norteamericanas y se obtuvieron de los repositorios públicos del Centro Nacional de Investigación Biotecnológica (NCBI). La serie restante es española y fue obtenida gracias a una colaboración con el Hospital Clinic de Barcelona. En total se emplearon para este estudio los genotipos obtenidos del ADN de 2697 individuos independientes (1305 casos de PD compleja y 1391 controles).  Unlike previous studies, the present invention uses the meta-analysis of three independent series of cases of complex PD and unrelated controls. Two of the series are North American and were obtained from the public repositories of the National Center for Biotechnological Research (NCBI). The remaining series is Spanish and was obtained thanks to a collaboration with the Hospital Clinic of Barcelona. In total, the genotypes obtained from the DNA of 2697 independent individuals (1305 cases of complex PD and 1391 controls) were used for this study.
- HFCC aplicado a PD.  - HFCC applied to PD.
La tecnología HFCC puede aplicarse para seleccionar un conjunto de marcadores genéticos diplotípicos (combinaciones de genotipos de dos SNPs independientes) para explicar el riesgo diferencial que tienen los individuos a padecer PD. Estos marcadores, aunque seleccionados por parejas, conservan el grado de asociación individualmente, por lo que pueden utilizarse tanto individualmente, como en parejas, o en conj unto . HFCC technology can be applied to select a set of diplomatic genetic markers (genotype combinations of two independent SNPs) to explain the differential risk that individuals have to suffer from PD. These markers, although selected in pairs, retain the degree of association individually, so they can be used both individually, as in pairs, or together.
Para seleccionar el conjunto de marcadores asociados a PD, se pueden utilizar los datos públicos de genomas completos. Los repositorios que custodian dichos datos, habrán sido los responsables de realizar el genotipado de las muestras de los individuos. La tecnología HFCC se aplica mediante la utilización de dispositivos o soportes sólidos que contienen oligonucleótidos capaces de hibridar con los SNPs conocidos. Estos dispositivos se conocen en el campo de la técnica como chips de SNPs o SNP Arrays y están disponibles comercialmente . Utilizando estos materiales de partida, puede explorarse el genoma de los individuos seleccionados para identificar todas las combinaciones diplotípicas posibles. En la aplicación HFCC se seleccionan los filtros adecuados. HFCC utiliza sistemáticamente tres filtros en diferentes fases del estudio: un filtro de ruido, que es una comparación control-control para eliminar asociaciones espúreas debidas a mala randomización del diplotipo en el grupo de controles, un filtro de direcciones que selecciona sóla aquellas combinaciones en las que la dirección del efecto es concordante y un filtro alelos tractores (tracking alíele filter) que identifica marcadores fuertemente desviados que introducen falsos positivos. Se incluyen, además, los parámetros estadísticos deseados, para lo cual el HFCC dispone de un algoritmo de computación paralela. La selección de los marcadores deseados, se efectúa automáticamente y posteriormente se ordenan y priorizan dichos SNPs bien utilizando un software adecuado a tal efecto o bien mediante estrategias de análisis condicional en función de genes previamente asociados a PD. De esta manera se pueden seleccionar las mejores parejas de SNPs para diseñar un ensayo de validación, que se realiza en series independientes para depurar la selección, eliminar falsos positivos y retener los SNPs consistentemente asociados a PD. To select the set of markers associated with PD, the public data of complete genomes can be used. The repositories that guard said data will have been responsible for genotyping the samples of the individuals. HFCC technology is applied through the use of solid devices or supports containing oligonucleotides capable of hybridizing with known SNPs. These devices are known in the art as SNPs or SNP Arrays chips and are commercially available. Using these starting materials, the genome of the selected individuals can be explored to identify all possible diplomotypic combinations. The appropriate filters are selected in the HFCC application. HFCC systematically uses three filters in different phases of the study: a noise filter, which is a control-control comparison to eliminate spurious associations due to bad randomization of the diploma in the control group, an address filter that selects only those combinations in the that the direction of the effect is concordant and a filter tractor alleles (tracking ally filter) that identifies strongly deviated markers that introduce false positives. In addition, the desired statistical parameters are included, for which the HFCC has a parallel computing algorithm. The selection of the desired markers is carried out automatically and subsequently, these SNPs are ordered and prioritized using either suitable software for this purpose or through conditional analysis strategies based on genes previously associated with PD. In this way, the best pairs of SNPs can be selected to design a validation test, which is carried out in series independent to purify the selection, eliminate false positives and retain the SNPs consistently associated with PD.
- Diseño de un ensayo para la validación de los SNPs seleccionados .  - Design of a trial for the validation of the selected SNPs.
Para validar los SNPs seleccionados, se determinan las secuencias génicas que han de sintetizarse para el ensayo de genotipación múltiple (Oligonucleotide Pooling Assay; OPA) . Estas secuencias deben ser aptas para su lectura simultánea en el Sistema BeadXpress. La tecnología BeadXpress (I Ilumina inc, EEUU) consiste un lector láser de dos canales simultáneos no confocales de resolución de 30nm que permite leer los cilindros hológráficos que se emplean en la tecnología Veracode (I Ilumina inc, EEUU) (véase a continuación) .  To validate the selected SNPs, the gene sequences to be synthesized for the multiple genotyping assay (Oligonucleotide Pooling Assay; OPA) are determined. These sequences must be suitable for simultaneous reading in the BeadXpress System. The BeadXpress (I Ilumina inc, USA) technology consists of a 30nm resolution two-channel simultaneous non-confocal laser reader that allows you to read the holographic cylinders used in Veracode technology (I Ilumina inc, USA) (see below).
- Genotipación (tecnología Veracode)  - Genotyping (Veracode technology)
Los SNPs de la invención pueden genotiparse mediante una muestra aislada de un individuo, poniendo en contacto sondas para los SNP con ADN genómico aislado de dicha muestra, bajo condiciones apropiadas para la hibridización de dicha sonda a dicho gen. Dicha sonda puede ser un ADN de tipo salvaje (es decir, el SNP que se investiga no está presente en la sonda, y la hibridización tiene lugar cuando el SNP según la invención no se encuentra en el ADN en la muestra) , o un mutante (es decir, transportando el SNP que se busca, y la hibridización sólo tiene lugar si el SNP se encuentra en el ADN en la muestra) . A efectos de la presente invención se entiende el término "muestra" como una porción de tejido o volumen de fluido corporal, preferentemente sangre periférica, pero que puede ser también cualquier otro tejido aislado del cuerpo, por ejemplo mediante una biopsia, o muestra de fluido corporal que contenga el ADN del individuo en estudio. Un experto en la materia conoce los procedimientos para determinar las sondas alélicas específicas a utilizar, sus longitudes, preferentemente de entre 50-300 bases, o las condiciones de hibridización . Pueden utilizarse secuencias que flanqueen al SNP y que el experto encontrará en el listado de secuencias de la presente invención. SNPs of the invention can be genotyped by an isolated sample of an individual, by contacting probes for SNPs with genomic DNA isolated from said sample, under appropriate conditions for hybridization of said probe to said gene. Said probe can be a wild-type DNA (that is, the SNP under investigation is not present in the probe, and hybridization takes place when the SNP according to the invention is not found in the DNA in the sample), or a mutant (that is, transporting the SNP that is sought, and hybridization only takes place if the SNP is found in the DNA in the sample). For the purposes of the present invention, the term "sample" is understood as a portion of tissue or volume of body fluid, preferably peripheral blood, but which can also be any other tissue isolated from the body, for example by biopsy, or fluid sample body that contains the DNA of the individual under study. One skilled in the art knows the procedures for determining the specific allelic probes to be used, their lengths, preferably between 50-300 bases, or the hybridization conditions. Sequences that flank the SNP can be used and that the expert will find in the sequence listing of the present invention.
Veracode es la tecnología de elección para la validación de resultados de HFCC. Esta tecnología emplea el poder del código holográfico digital para ofrecer un sistema de detección robusto y es ideal para múltiples ensayos que requieren alta precisión, reproducibilidad y rapidez. El sistema emplea unas microesferas diferenciadas por una marca holográfica de alta densidad (24-bit) . Cuando las microesferas VeraCode son excitadas mediante un láser, cada una emite un código de imagen único, lo que permite una detección rápida y específica por el sistema de lectura denominado "BeadXpress Reader System" ( Illumina inc, EEUU). De acuerdo con el nivel de multiplex requerido, los ensayos son creados mediante la mezcla de diferentes micro-esferas (cilindros) con distintos códigos, que pueden ir desde uno a varios cientos. Cada tipo de micro-esfera lleva en la superficie unida covalentemente especies de oligonucleótidos específicos de la región que contiene el polimorfismo. Previamente dicha región es detectada en cada uno de los genomas a estudiar mediante el sistema GoldenGate de Illumina (Illumina Inc, EEUU). De esta forma el sistema puede interrogar simultáneamente hasta 384 polimorfismos de un único nucleótido (SNP) por muestra de ADN . Brevemente, el sistema Goldengate permite la genotipación de SNPs mediante un proceso de extensión y ligación del ADN "secuencia- específico". El sistema permite ligar sólo aquellos oligos hibridados correctamente. Los fragmentos correctos, una vez ligados, son amplificados con primers universales. En el —* t — — - - -Veracode is the technology of choice for the validation of HFCC results. This technology uses the power of digital holographic code to offer a robust detection system and is ideal for multiple tests that require high accuracy, reproducibility and speed. The system uses microspheres differentiated by a holographic mark of high density (24-bit). When the VeraCode microspheres are excited by a laser, each one emits a unique image code, which allows a quick and specific detection by the reading system called "BeadXpress Reader System" (Illumina inc, USA). According to the level of multiplex required, the tests are created by mixing different microspheres (cylinders) with different codes, which can range from one to several hundred. Each type of micro-sphere carries on the surface covalently bound oligonucleotide species specific to the region containing the polymorphism. Previously, said region is detected in each of the genomes to be studied using the Illumina GoldenGate system (Illumina Inc, USA). In this way, the system can simultaneously interrogate up to 384 single nucleotide polymorphisms (SNPs) per DNA sample. Briefly, the Goldengate system allows the genotyping of SNPs through a process of extension and ligation of "sequence-specific" DNA. The system allows to link only those oligos hybridized correctly. The correct fragments, once bound, are amplified with universal primers. At - * t - - - - -
14 sistema GoldenGate el ADN problema se activa mediante una reacción química con la biotina. Después se elimina el exceso de biotina mediante una purificación en columna. Para iniciar el ensayo, la OPA (tabla 1) que contiene los oligonucleotidos se añade y se efectúa una hibridación con el ADN activado con la biotina. La mezcla de ADN activado-OPA se liga a unas partículas paramagnéticas conjugadas con estreptavidina (SA- P Ps ) . Una vez finalizada la hibridación se procede a la eliminación del exceso de oligonucleotidos no hibridados y los mal apareados. El siguiente paso es la realización de la extensión y ligación alelo-especí fica que produce un molde sintético de cada alelo detectado. Estos moldes se someten a una reacción de PCR con primers universales fluorescentes que contienen unos códigos de secuencias reconocibles por los cilindros holográficos que detecta el BeadXpress. La cadena fluorescente del producto de PCR se aisla y se híbrida a los cilindros holográficos. Como conclusión, después de un proceso de lavado, los cilindros hibridados se escanean en el sistema y se analizan los resultados con el software BeadStudio (Illumina Inc, EEUU) . La interpretación de los resultados y la anotación de los genotipos obtenidos también se realizan con el software BeadStudio (Illumina). 14 GoldenGate system the problem DNA is activated by a chemical reaction with biotin. The excess biotin is then removed by column purification. To start the assay, the OPA (table 1) containing the oligonucleotides is added and hybridization is performed with the biotin activated DNA. The mixture of activated DNA-OPA binds to paramagnetic particles conjugated with streptavidin (SA-P Ps). Once the hybridization is finished, the excess of unhybridized oligonucleotides and the poorly matched ones are eliminated. The next step is the realization of the allele-specific extension and ligation that produces a synthetic mold of each detected allele. These molds undergo a PCR reaction with universal fluorescent primers containing sequence codes recognizable by the holographic cylinders detected by the BeadXpress. The fluorescent chain of the PCR product is isolated and hybridized to the holographic cylinders. In conclusion, after a washing process, the hybridized cylinders are scanned in the system and the results are analyzed with the BeadStudio software (Illumina Inc, USA). The interpretation of the results and the annotation of the genotypes obtained are also carried out with the BeadStudio software (Illumina).
Empleando la tecnología descrita y dos series de validación, la presente invención provee un total de 36 nuevos SNPs (de ahora en adelante SNPs de la invención) útiles para predecir la predisposición a padecer PD compleja Tabla 1). Por lo tanto, un primer objeto de la presente invención es un procedimiento in Vitro para determinar la predisposición a padecer la enfermedad de Parkinson en una muestra aislada de un individuo, que comprende el análisis genotípico de al menos un SNP a elegir del grupo formado por: rs577562, rs2986574, rsllll9097, rs4658673, rs4490160, rsl0496075, rsl2987286, rs847126, rs9036, rs6805546, rs4505714, rs7650598, rsl0155062, rs218280, rs869026, rs3094694, rsl549355, rs7747934, rs4583999, rsl0945364, rsll56143, rsl888349, rsl2353255, rs7027515, rs332185, rs763373, rsl0750861, rsll564173, rs2332631, rsl039189, rs3936139, rs8109493, rs761267, rs6518223, rsl364658, rs353111, y combinaciones de los mismos. Using the described technology and two validation series, the present invention provides a total of 36 new SNPs (hereinafter SNPs of the invention) useful for predicting the predisposition to suffer complex PD Table 1). Therefore, a first object of the present invention is an in vitro method for determining the predisposition to suffer from Parkinson's disease in an isolated sample of an individual, comprising genotypic analysis of at least one SNP to be chosen from the group consisting of : rs577562, rs2986574, rsllll9097, rs4658673, rs4490160, rsl0496075, rsl2987286, rs847126, rs9036, rs6805546, rs4505714, rs7650598, rsl0155062, rs218280, rs869026, rs3094694, rsl549355, rs7747934, rs4583999, rsl0945364, rsll56143, rsl888349, rsl2353255, rs7027515, rs332185, rs763373, rsl0750861, rsll564173, rs2332631, rsl039189, rs3936139, rs8109493, rs761267, rs6518223, rsl364658, rs353111, and combinations thereof.
Tabla 1. Relación entre los SNPs de la invención y la lista de secuencias de la invención. Table 1. Relationship between the SNPs of the invention and the sequence list of the invention.
SNP Variación (símbolo SEQ ID No  SNP Variation (symbol SEQ ID No
En el listado de secuencias)  In the sequence listing)
rs577562 [A/G] (r) 1  rs577562 [A / G] (r) 1
rs2986574 [A/G] (r) 2  rs2986574 [A / G] (r) 2
rsllll9097 [A/G] (r) 3  rsllll9097 [A / G] (r) 3
rs4658673 [A/G] (r) 4  rs4658673 [A / G] (r) 4
rs4490160 [A/G] (r) 5  rs4490160 [A / G] (r) 5
rsl0496075 [T/C] (y) 6  rsl0496075 [T / C] (y) 6
rsl2987286 [T/G] (k) 7  rsl2987286 [T / G] (k) 7
rs847126 [T/C] (y) 8  rs847126 [T / C] (y) 8
rs9036 [T/C] (y) 9  rs9036 [T / C] (y) 9
rs6805546 [T/G] (k) 10  rs6805546 [T / G] (k) 10
rs4505714 [A/G] (r) 11  rs4505714 [A / G] (r) 11
rs7650598 [A/G] (r) 12  rs7650598 [A / G] (r) 12
rsl0155062 [A/G] (r) 13  rsl0155062 [A / G] (r) 13
rs218280 [T/G] (k) 14  rs218280 [T / G] (k) 14
rs869026 [T/G] (k) 15  rs869026 [T / G] (k) 15
rs3094694 [A/G] (r) 16  rs3094694 [A / G] (r) 16
rsl549355 [T/G] (k) 17  rsl549355 [T / G] (k) 17
rs7747934 [T/C] (y) 18  rs7747934 [T / C] (y) 18
rs4583999 [A/G] (r) 19  rs4583999 [A / G] (r) 19
rsl0945364 [T/C] (y) 20  rsl0945364 [T / C] (y) 20
rsll56143 [T/C] (y) 21  rsll56143 [T / C] (y) 21
rsl888349 [T/G] (k) 22  rsl888349 [T / G] (k) 22
rsl2353255 [A/G] (r) 23  rsl2353255 [A / G] (r) 23
rs7027515 [T/C] (y) 24  rs7027515 [T / C] (y) 24
rs332185 [T/C] (y) 25  rs332185 [T / C] (y) 25
rs763373 [C/G] (s) 26  rs763373 [C / G] (s) 26
rsl0750861 [T/C] (y) 27  rsl0750861 [T / C] (y) 27
rsll564173 [A/G] (r) 28  rsll564173 [A / G] (r) 28
rs2332631 [T/G] (k) 29 SNP Variación (símbolo SEQ ID No rs2332631 [T / G] (k) 29 SNP Variation (symbol SEQ ID No
En el listado de secuencias)  In the sequence listing)
rsl039189 [A/C] (m) 30  rsl039189 [A / C] (m) 30
rs3936139 [T/C] (y) 31  rs3936139 [T / C] (y) 31
rs8109493 [T/C] (y) 32  rs8109493 [T / C] (y) 32
rs761267 [A/C] (m) 33  rs761267 [A / C] (m) 33
rs6518223 [T/C] (y) 34  rs6518223 [T / C] (y) 34
rsl364658 [C/G] (s) 35  rsl364658 [C / G] (s) 35
rs353111 [T/G] (k) 36  rs353111 [T / G] (k) 36
La Tabla 2 muestra la localización cromosómica, el nombre del gen y el número de acceso de los SNPs de la invención mientras que en la Tabla 1 se muestra la relación entre los SNPs de la invención, la variación que presentan, y la lista de secuencias de la invención, que incluyen las secuencias nucleotídicas que los flanquean. Table 2 shows the chromosomal location, the name of the gene and the access number of the SNPs of the invention while in Table 1 the relationship between the SNPs of the invention, the variation they present, and the sequence list are shown. of the invention, which include the flanking nucleotide sequences.
En una realización preferente, el procedimiento in Vitro de la invención es útil para predecir el riesgo de padecer PD compleja a partir de una muestra aislada de un individuo.  In a preferred embodiment, the in vitro method of the invention is useful for predicting the risk of complex PD from an isolated sample of an individual.
En otra realización preferente, el procedimiento in Vitro de la invención utiliza los SNPs rs869026 y rsl0945364, situados en los cromosomas 5 y 6 respectivamente. Según este método, cuando el genotipo obtenido de la muestra aislada de un individuo presenta homocigosis de alelo T de rs869026 combinado con la presencia en heterocigosis del alelo C de rsl0945364 (diplotipo TT/CT de los marcadores), dicho individuo tiene un 66-72% más de riesgo de padecer la enfermedad de Parkinson respecto a los no portadores (Tabla 3) .  In another preferred embodiment, the in vitro method of the invention uses the SNPs rs869026 and rsl0945364, located on chromosomes 5 and 6 respectively. According to this method, when the genotype obtained from the isolated sample of an individual presents homozygosis of the T allele of rs869026 combined with the presence in heterozygous of the C allele of rsl0945364 (TT / CT diploma of the markers), said individual has a 66-72 % more risk of suffering from Parkinson's disease compared to non-carriers (Table 3).
El análisis detallado del grado de asociación de los SNPs individuales con la enfermedad, resultó en la identificación de 14 nuevos SNPs con una probabilidad de asociación altamente significativa (Ejemplo 3). Por lo tanto otro aspecto de la presente invención provee de un procedimiento in vitro para determinar la predisposición a padecer la enfermedad de Parkinson en una muestra aislada de un individuo, que comprende el análisis genotipico de al menos un SNP a elegir del grupo formado por: rsl039189, rsl2987286, rsl364658, rsl549355, rs218280, rs2332631, rs353111, rs3936139, rs4490160, rs577562 rs6805546, rs761267, rs7650598, rs869026 y sus combinaciones. The detailed analysis of the degree of association of individual SNPs with the disease resulted in the identification of 14 new SNPs with a highly significant probability of association (Example 3). Therefore another aspect of the present invention provides an in vitro method for determining the predisposition to suffer from Parkinson's disease in an isolated sample of an individual, which comprises the genotypic analysis of at least one SNP to choose from the group consisting of: rsl039189, rsl2987286, rsl364658, rsl549355, rs218280, rs2332631, rs353111, rs394475169, rs3957751606 , rs761267, rs7650598, rs869026 and their combinations.
Una manera diferente de demostrar la capacidad diagnóstica de los SNPs de la invención es la elaboración de un panel de marcadores (SNPs) con capacidad predictiva. Para la construcción de dicho panel, se emplea una combinación de SNPs asociados al "status" de casos (estos son los marcadores presentes en la Tabla 5) a los que se añade, además, un pequeño subconjunto de marcadores que presentan indicios estadísticamente significativos de asociación con la edad de comienzo de la enfermedad (ΑΆ.Ο, age-at-onset ) (Tabla 6) . El ejemplo 4 muestra la creación de dicho panel predictivo, que puede utilizarse como un sistema de puntuación genético basado en los genotipos de los loci, que permite la clasificación de individuos afectados de PD compleja sin que haga falta tener en cuenta ningún otro factor.  A different way of demonstrating the diagnostic capacity of the SNPs of the invention is the development of a panel of markers (SNPs) with predictive capacity. For the construction of said panel, a combination of SNPs associated with the "status" of cases is used (these are the markers present in Table 5) to which is also added a small subset of markers that present statistically significant indications of association with the age of onset of the disease (ΑΆ.Ο, age-at-onset) (Table 6). Example 4 shows the creation of said predictive panel, which can be used as a genetic scoring system based on the genotypes of the loci, which allows the classification of affected individuals of complex PD without having to take into account any other factor.
Según lo anterior, en una realización preferente, de la presente invención es un procedimiento in Vitro basado en un sistema de puntuación genético que comprende el análisis genotipico conjunto de los SNPs: rs577562, rs2986574, rsllll9097, rs4658673, rs4490160, rsl0496075, rsl2987286, rs847126, rs9036, rs6805546, rs4505714, rs7650598, rsl0155062, rs218280, rs869026, rs3094694, rsl549355, rs7747934, rs4583999, rsl0945364, rsll56143, rsl888349, rsl2353255, rs7027515, rs332185, rs763373, rsl0750861, rsll564173, rs2332631, rsl039189, rs3936139, rs8109493, rs761267, rs6518223 y sus combinaciones, preferentemente, la combinación de todos ellos. Con el objeto de analizar conjuntamente los SNPs de la invención en un ensayo múltiple, es también objeto de la presente invención un conjunto de reactivos o kit para la determinación de la predisposición a padecer la enfermedad de parkinson en una muestra aislada de un individuo, caracterizado porque comprende un ácido nucleico que detecta al menos un SNP a elegir del grupo formado por: rs577562, rs2986574, rsllll9097, rs4658673, rs4490160, rsl0496075, rsl2987286, rs847126, rs9036, rs6805546, rs4505714, rs7650598, rsl0155062, rs218280, rs869026, rs3094694, rsl549355, rs7747934, rs4583999, rsl0945364, rsll56143, rsl888349, rsl2353255, rs7027515, rs332185, rs763373, rsl0750861, rsll564173, rs2332631, rsl039189, rs3936139, rs8109493, rs761267, rs6518223, rsl364658, rs353111, y combinaciones de éstos, preferentemente, la combinación todos ellos. According to the above, in a preferred embodiment, of the present invention is an in vitro method based on a genetic scoring system comprising the joint genotypic analysis of the SNPs: rs577562, rs2986574, rsllll9097, rs4658673, rs4490160, rsl0496075, rsl2987126 , rs9036, rs6805546, rs4505714, rs7650598, rsl0155062, rs218280, rs869026, rs3094694, rsl549355, rs7747934, rs4583999, rsl0945364, rsll56143, rsl888349, rsl2353255, rs7027515, rs332185, rs763373, rsl0750861, rsll564173, rs2332631, rsl039189, rs3936139, rs8109493, rs761267 , rs6518223 and their combinations, preferably, the combination of all of them. In order to jointly analyze the SNPs of the invention in a multiple assay, a set of reagents or kit for the determination of the predisposition to suffer from parkinson's disease in an isolated sample of an individual, characterized is also the subject of the present invention. because it comprises a nucleic acid that detects at least one SNP to be chosen from the group consisting of: rs577562, rs2986574, rsllll9097, rs4658673, rs4490160, rsl0496075, rsl2987286, rs847126, rs9036, rs6805546, rs45029062629029022629029029029022629029029021602, 09021602, 09021602, 0902302146 rsl549355, rs7747934, rs4583999, rsl0945364, rsll56143, rsl888349, rsl2353255, rs7027515, rs332185, rs763373, rsl0750861, rsll564173, rs2332631, rsl039189, rs3936139, rs8109493, rs761267, rs6518223, rsl364658, rs353111, and combinations thereof, preferably the combination all they.
Ejemplo 1. HFCC aplicado a PD Example 1. HFCC applied to PD
Para llevar a cabo la presente invención se utilizaron datos de genoma completo públicos que se obtuvieron de los repositorios del centro de recursos de SNPs, ADN y lineas celulares que el Instituto Nacional de Investigación de Enfermedades Neurológicas de los Estados Unidos (NINDS) mantiene en la empresa Coriell (http://ccr.coriell.org/ninds, datos consultados en Diciembre de 2006) . El genotipado de las muestras empleadas para realizar HFCC se efectuó en el Instituto Nacional de Envejecimiento (NIA, Bethesda, MD, EEUU) (35) . El HFCC se realizó con genotipos de 270 casos no relacionados de PD idiopático y 271 controles poblacionales normales desde un punto de vista neurológico y no relacionados entre ellos, ni con los controles. Los 541 individuos disponibles se genotiparon para 396.591 SNPs diferentes distribuidos en los 22 autosomas. Para ello se emplearon dos chips (SNP arrays Infinium I y II) de la compañía Illumina (Illumina Inc, EEUU) . Con estos datos de partida se utilizó la tecnología HFCC para explorar todas las combinaciones diplotípicas posibles de los 396.591 marcadores SNPs genotipados. Se efectuó, en consecuencia, un rastreo exhaustivo de todos los patrones diplotípicos existentes. De esta manera, se divide la base de datos disponible en tres grupos de replicación de 90 casos y 90 controles (una de ellas llevaba un control más) y se ejecutó el software HFCC programando la evaluación estadística de todos los diplotipos (modelos 2-loci M1,M2 y Mi 6 de acuerdo con Li y Reich 2000 (7) ) . La evaluación de estos nueve modelos genéticos, correspondientes a los diplotipos, supone el cálculo de unos 78,6 billones de test estadísticos (Test de Wald ; S . D. Silvey "The Lagrangian Multiplier Test," Annals of Mathematical Statistics, 30, (1959), pp. 389-407. para lo que se emplea el algoritmo de computación paralela programado en HFCC (36) . El punto de corte para pre-seleccionar diplotipos candidatos se estableció en un Test de Wald mayor o igual a 6,64 puntos que equivale, aproximadamente, a un valor p<0,01. Es importante destacar que este valor ha de ser alcanzado en los tres grupos de replicación anteriormente mencionados. Con lo cual los positivos suministrados por HFCC tienen un valor de p "agregado" (valor de significación empleado en HFCC: se calcula usando el punto de corte establecido para cada grupo de comparación y estrato elevado al número de grupos de comparación empleados durante el estudio) de p~0, 000001 o inferior . In order to carry out the present invention, public complete genome data were used that were obtained from the repositories of the SNPs, DNA and cell lines resource center that the National Institute of Neurological Diseases Research of the United States (NINDS) maintains in the Coriell company (http://ccr.coriell.org/ninds, data consulted in December 2006). Genotyping of the samples used to perform HFCC was carried out at the National Institute of Aging (NIA, Bethesda, MD, USA) (35). The HFCC was performed with genotypes of 270 unrelated cases of idiopathic PD and 271 normal population controls from a neurological point of view and not related to each other, nor with the controls. The 541 available individuals were genotyped for 396,591 different SNPs distributed in the 22 autosomes. To do this they used two chips (SNP arrays Infinium I and II) of the Illumina company (Illumina Inc, USA). With this starting data, HFCC technology was used to explore all possible diplomotypic combinations of the 396,591 genotyped SNPs markers. As a result, an exhaustive screening of all existing diplomatic patterns was carried out. In this way, the database available is divided into three replication groups of 90 cases and 90 controls (one of them had one more control) and the HFCC software was executed programming the statistical evaluation of all the diplomas (2-loci models M1, M2 and Mi 6 according to Li and Reich 2000 (7)). The evaluation of these nine genetic models, corresponding to diplomas, involves the calculation of some 78.6 billion statistical tests (Wald Test; S. Silvey "The Lagrangian Multiplier Test," Annals of Mathematical Statistics, 30, ( 1959), pp. 389-407, for which the parallel computing algorithm programmed in HFCC is used (36) The cut-off point to pre-select candidate diplomas was established in a Wald Test greater than or equal to 6.64 points equivalent to approximately p <0.01 It is important to note that this value has to be reached in the three aforementioned replication groups, whereby the positives provided by HFCC have an "aggregate" p value (significance value used in HFCC: it is calculated using the cut-off point established for each comparison group and stratum raised to the number of comparison groups used during the study) of p ~ 0.000001 or less.
Después de aplicar los filtros de HFCC, el sistema seleccionó un 4,4% de los marcadores (17825 SNPs) que formaban 26371 combinaciones diplotípicas. Éstas se ordenaron y priorizaron empleando bien el software Alambique (21) o bien estrategias de análisis condicional (buscando parejas de marcadores que al menos contuviesen SNPs dentro de genes previamente asociados a PD) . Los 384 SNPs seleccionados más prometedores (tabla 2) fueron correspondientemente validados en series independientes (ver más adelante) . After applying the HFCC filters, the system selected 4.4% of the markers (17825 SNPs) that formed 26371 diploma combinations. These were ordered and prioritized using either Alambique software (21) or conditional analysis strategies (looking for pairs of markers that at least contained SNPs within genes previously associated with PD). The 384 most promising selected SNPs (table 2) were correspondingly validated in independent series (see below).
Tabla 2. SNPs seleccionados mediante HFCC. Datos de la cadena sentido o "forward". CV: clase de validación; BV: Bandeja de validación, LGR: Localización Relativa al Gen. Table 2. SNPs selected by HFCC. Sense or forward chain data. CV: validation class; BV: Validation tray, LGR: Location Relating to Gen.
SNP Cr Coord CV BV Gen Nombre Gen Número de Acceso Localización LRG rsl003167 6 43963001 3 goldengate 7422 VEGFA NM_001033756.1 adyacente a_3UTR -100799 rsl0046663 8 2983419 3 goldengate 64478 CSMD1 NM_033225.3 intron -3975 rsl0O53056 5 96069176 2 two-hit or HapMap 831 CAST NM_173060.1 intron -4801 rsl0078721 5 92446445 2 two-hit or HapMap 391811 LOC391811 XR_016127.1 adyacente a_5UTR -182298 rsl008577 19 46879134 2 two-hit or HapMap 1087 CEACAM7 NM_006890.1 intron -422 rsl0094702 8 89403625 3 goldengate 4325 MMP16 NM_005941.3 intron -4795 rsl0096279 8 3212285 2 two-hit or HapMap 64478 CSMD1 NM_033225.3 intron -109 rsl0121215 9 26159347 3 goldengate 441390 -99 NM_001004352.1 adyacente a_3UTR -50941 rsl0155062 4 7184806 2 two-hit or HapMap 728529 LOC728529 XM_001127674.1 adyacente a_3UTR -55088 rsl0201616 2 75529368 3 goldengate 84141 TMEM166 NM_032181.1 adyacente a_3UTR -43584 rsl022242 12 69492622 2 two-hit or HapMap 5801 PTP NM_002849.2 intron -47797 rsl035316 2 224848397 3 goldengate 79843 FAM124B NM_024785.2 adyacente a_3UTR -103262 rsl0496075 2 57762864 2 two-hit or HapMap 7444 VR 2 NM_006296.3 adyacente a_5UTR -364360 rsl0511001 3 70715884 2 two-hit or HapMap 401072 -99 NM_001013679.1 adyacente a_3UTR -29555 rsl0511680 9 20277623 2 two-hit or HapMap 4300 MLLT3 NM_004529.1 adyacente a_3UTR -57345 rsl0512174 9 88076394 3 goldengate 81689 ISCA1 NM_030940.3 intron -348 rslOS 16970 4 96529259 2 two-hit or HapMap 8633 UNC5C NM_003728.2 intron -53454 rsl060545 9 116861439 3 goldengate 3371 TNC NM_002160.1 intron -391 rsl0747874 12 59144947 2 two-hit or HapMap 390335 LOC390335 XM_0O1129525.1 adyacente a_3UTR -652323 rsl0750861 11 56937983 2 two-hit or HapMap 29015 SLC43A3 NM_017611.2 intron -681 rsl0796886 1 36787398 3 goldengate 1441 CSF3R NM_172313.1 adyacente a_5UTR -66302 rsl0797007 1 156562253 3 goldengate 910 CD1B NM_001764.1 adyacente a_3UTR -2150 rsl0801705 1 89272970 3 goldengate 2635 GBP3 NM_018284.1 adyacente a_5UTR -11838 rsl0853282 18 2372165 3 goldengate 64863 METTL4 NM_022840.2 adyacente a_3UTR -155364 rsl0916407 1 227216444 3 goldengate 677790 TMEM78 NM_001040079.1 adyacente a_5UTR -235562 rsl0935844 3 152611585 3 goldengate 116931 MED12L NM_053002.3 intron -171 rsl0945364 6 169193728 3 goldengate 7058 THBS2 NM_003247.2 adyacente a_3UTR -164072 rsll008264 10 31129838 3 goldengate 220929 ZNF438 NM_182755.1 adyacente a_3UTR -43731 rsll06145 3 87631095 3 goldengate 643766 LOC643766 XR_016476.1 adyacente a_3UTR -131046 rsll081496 18 9716631 2 two-hit or HapMap 11031 RAB31 NM_006868.2 intron -18190 rsll088449 21 39107267 2 two-hit or HapMap 2114 ETS2 NMJ305239.4 intron -359 rsllll9097 1 206813021 3 goldengate 5362 PLXNA2 NM_025179.2 adyacente a_5UTR -329282 rslll96543 10 115684622 2 two-hit or HapMap 374354 NHLRC2 NM_198514.2 adyacente a_3UTR -26180 rsl 14305 12 5118252 3 goldengate 3741 KCNA5 NM_002234.2 adyacente a_3UTR -92042 rsll50996 12 31960144 2 two-hit or HapMap 55196 C12orf35 NM_018169.2 adyacente a_5UTR -43476 rsll564252 12 39100000 2 two-hit or HapMap 283463 MUC19 XM_497341.3 ntron •276 SNP Cr Coord CV BV Gen Nombre Gen Número de Acceso Localización LRG rsll655589 17 58961895 2 two-hit or HapMap 81033 KCNH6 NM_173092.1 intron -260 rsll674954 2 1262038 3 goldengate 54221 SNTG2 NM_018968.2 adyacente a_3UTR -8114 rsll691934 2 86018954 3 goldengate 8869 ST3GAL5 NM_003896.2 adyacente a_5UTR -49306 rsll694263 2 237855128 2 two-hit or HapMap 728245 LOC728245 XM_001126970.1 adyacente a_5UTR -14194 rsll715563 3 83873910 3 goldengate 643665 LOC643665 XM_931729.2 adyacente a_5UTR -1214935 rsll738500 5 114525871 2 two-hit or HapMap 55521 TRIM36 NM_018700.3 intron -1243 rsll860440 16 1697619 2 two-hit or HapMap 23162 MAPK8IP3 NM_001040439.1 intron -960 rsll893869 2 106032330 3 goldengate 84417 C2orf40 NM_032411.1 adyacente a_5UTR -16215 rsl203638 1 13994479 3 goldengate 7799 PRDM2 NM_015866.3 intron -7320 rsl2081567 1 151467611 3 goldengate 391102 LOC391102 XR_016996.1 adyacente a_3UTR -2505 rsl2119878 1 9141308 2 two-hit or HapMap 727721 LOC727721 XM_001123441.1 adyacente a_3UTR -17730 rsl2144940 1 99705926 3 goldengate 9890 LPPR4 NM_014839.3 adyacente a_3UTR -158198 rsl2353255 9 20911332 3 goldengate 54914 KIAA1797 NM_017794.2 intron -2327 rsl2412945 10 54468113 2 two-hit or HapMap 4153 MBL2 NMJ 00242.1 adyacente a_5UTR -266647 rsl2434638 14 89679833 2 two-hit or HapMap 56659 KCNK13 NM_022054.2 intron -40375 rsl2495688 3 147525535 3 goldengate 440981 LOC440981 XM_496663.3 adyacente a_3UTR -66302 rsl2520264 5 7698474 2 two-hit or HapMap 108 ADCY2 NM_020546.1 intron -19045 rsl2575639 11 109869510 2 two-hit or HapMap 2230 FDX1 NM_004109.3 adyacente a_3UTR -28695 rsl2672177 7 101813787 3 goldengate 79706 PR RIP1 NM_024653.1 adyacente a_5UTR -10028 rsl267658 15 81011209 2 two-hit or HapMap 64506 CPEB1 NM_030594.2 intron -702 rsl2929690 16 81693979 2 two-hit or HapMap 1012 CDH13 NM_001257.3 intron -22512 rsl2976749 19 43811334 2 two-hit or HapMap 27335 EIF3K NM_013234.2 intron -2752 rsl2987286 2 148380038 3 goldengate 92 ACVR2A NM_001616.3 intron -6101 rsl329580 9 81776189 3 goldengate 347119 LOC347119 XM_928399.1 adyacente a_3UTR -105858 rsl364658 2 148324278 3 goldengate 92 ACVR2A NM_001616.3 intron -5032 rsl365461 18 34356804 3 goldengate 388474 LOC388474 XM_371115.5 adyacente a_3UTR -811939 rsl366545 16 76022537 2 two-hit or HapMap 170692 ADAMTS18 NM_199355.1 intron -156 rsl370699 2 86025785 3 goldengate 8869 ST3GAL5 NM_003896.2 adyacente a_5UTR -56137 rsl385331 3 197835612 3 goldengate 375387 LRRC33 NM_198565.1 adyacente a_5UTR -15441 rsl397053 11 56267270 2 two-hit or HapMap 283189 OR9G4 NM_001005284.1 coding [390/593] rsl447665 3 127444361 3 goldengate 644662 LOC644662 XM_933903.2 adyacente a_5UTR -23968 rsl480691 8 14808735 2 two-hit or HapMap 137868 SGCZ NM_139167.2 intron -330730 rsl499005 3 20895844 3 goldengate 728976 LOC728976 XM_001128967.1 adyacente a_3UTR -23063 rsl50858 17 3479419 2 two-hit or HapMap 23729 CARKL NM_013276.2 intron -829 rsl511241 11 78746402 2 two-hit or HapMap 729746 LOC729746 XM_00U31186.1 adyacente a_5UTR -281322 rsl521937 2 53515720 3 goldengate 51130 ASB3 NM_145863.1 adyacente a_3UTR -234902 rsl527101 12 77063452 2 two-hit or HapMap 89795 NAV3 NM_014903.3 intron -828 rsl537685 10 116894953 3 goldengate 26033 ATRNL1 NM_207303.1 intron -14838 rsl549355 6 124849888 3 goldengate 154215 NKAIN2 NM_001040214.1 intron -131696 rsl573465 21 34871068 3 goldengate 1827 RCAN1 NM_203417.1 intron -37379 rsl62216 3 7833048 2 two-hit or HapMap 2917 GRM7 NM_000844.2 adyacente a_3UTR -74831 rsl667059 2 2925338 3 goldengate 729897 LOC729897 XM_001134159.1 adyacente a_3UTR -189426 rsl675247 18 3608506 3 goldengate 649446 FU35776 NM_001039796.1 adyacente a_3UTR -20156 rsl708694 18 32321976 3 goldengate 80206 FHOD3 NMJ325135.2 intron -13917 rsl725265 1 7480780 2 two-hit or HapMap 23261 CAMTA1 NM_015215.1 intron -30232 rsl7714109 4 28900684 2 two-hit or HapMap 645641 LOC645641 XM_928656.1 adyacente a_5UTR -466370 SNP Cr Coord CV 1 BV Gen Nombre Gen Número de Acceso Localización LRG rsl7740213 8 24430274 2 two-hit or HapMap 8756 ADAM7 NM_003817.1 adyacente a_3UTR -8103 rsl78214 14 26015206 2 two-hit or HapMap 4857 NOVA1 NM_006491.2 intron -3769 rsl792358 11 92040229 2 two-hit or HapMap 120114 FAT3 XM_926199.2 intron -29969 rsl828034 4 148309936 3 goldengate 83894 TTC29 NMJ331956.1 adyacente a_5UTR -223497 rsl836726 2 211892263 3 goldengate 2066 ERBB4 NM_005235.1 adyacente a_3UTR -62797 rsl861960 7 154977896 2 two-hit or HapMap 285888 CNPY1 XM_001129537.1 intron -1900 rsl874404 6 165012190 3 goldengate 728275 LOC728275 XM_001127046.1 adyacente a_3UTR -333408 rsl887279 1 182176783 3 goldengate 23127 GLT25D2 NM_015101.2 intron -239 rsl888349 7 138314672 2 two-hit or HapMap 92092 ZC3HAV1L NM_080660.2 adyacente a_3UTR -46320 rsl898111 15 45679590 2 two-hit or HapMap 80031 SEMA60 NM_020858.1 adyacente a_5UTR -118388 rsl901818 16 79761626 2 two-hit or HapMap 114780 PKD1L2 NM_052892.2 intron -423 rsl931704 10 129229799 3 goldengate 1793 DOCK1 NM_001380.2 adyacente a_3UTR -89029 rsl940814 18 14061989 3 goldengate 162655 ZNF519 NM_145287.1 adyacente a_3UTR -32735 rsl952198 14 69409508 2 two-hit or HapMap 64093 SMOC1 NM_022137.4 adyacente a_5UTR -6388 rsl953866 14 51387587 3 goldengate 54331 GNG2 NM_053064.2 adyacente a_5UTR -26473 rsl962293 9 2100744 3 goldengate 6595 SMARCA2 NM_139045.2 intron -327 rsl979541 6 159025401 3 goldengate 94120 SYTL3 NM_001009991.1 intron -1392 rs2011207 3 104449957 3 goldengate 644681 LOC644681 XM_927781.2 adyacente a_3UTR -290967 rs2026690 13 46381178 2 two-hit or HapMap 3356 HTR2A NM_000621.2 adyacente a_5UTR -13002 rs2041611 18 14077956 3 goldengate 162655 ZNF519 NM_145287.1 adyacente a_3UTR -16768 rs2047214 4 11150840 3 goldengate 9957 HS3ST1 NM_005114.2 adyacente a_5UTR -111205 rs2053380 7 12367362 2 two-hit or HapMap 221806 FU14712 XM_371878.5 coding [64/40] rs205752S 13 92755817 3 goldengate 10082 GPC6 NM_005708.2 intron -77947 rs2063239 12 81798372 3 goldengate 160335 TMTC2 NM_152588.1 intron -15356 rs2070862 17 1595044 2 two-hit or HapMap 5345 SERPINF2 NM_000934.1 coding [4/58] rs207620 13 32011403 3 goldengate 10443 RP11-298P3.3 NM_014887.1 adyacente a_5UTR -484 rs2077066 1 23650949 3 goldengate 55616 DDEFL1 NM_017707.2 intron -341 rs2113447 18 51827201 3 goldengate 642484 FU45743 NM_001039759.1 intron -5068 rs2129530 11 11354861 2 two-hit or HapMap 374378 GALNTL4 NM_198516.1 intron -444 rs214506 6 18329450 3 goldengate 221656 AOF1 NM_153042.2 intron -669 rs2163307 8 4293028 2 two-hit or HapMap 64478 CSMD1 NM_033225.3 intron -28033 rs218280 4 55119046 3 goldengate 3815 KIT NM_000222.1 adyacente a_5UTR -99872 rs2219925 18 47717831 3 goldengate 1630 DCC NM_005215.1 adyacente a_5UTR -403325 rs2220971 13 96826076 2 two-hit or HapMap 10150 MBNL2 NM_144778.2 intron -9268 rs222717 14 23161964 2 two-hit or HapMap 10202 DHRS2 NM_005794.2 adyacente a_5UTR -13458 rs2233568 9 35947355 3 goldengate 56656 OR2S2 NM_019897.1 coding [219/710] rs2235490 16 1683512 2 two-hit or HapMap 90861 HN1L NM_144570.2 intron -1525 rs2235616 20 48002534 2 two-hit or HapMap 55905 ZNF313 NM_018683.3 3UTR [448/1295] rs2241780 3 14495168 2 two-hit or HapMap 6533 SLC6A6 NM_003043.2 ntron -88 rs2242330 4 58129844 2 two-hit or HapMap 26228 STAP1 NM_012108.1 ntron -60 rs2242383 12 75962738 2 two-hit or HapMap 144455 E2F7 NM_203394.1 ntron -32 rs2253729 7 139589358 2 two-hit or HapMap 30853 HDM1D NM_030647.1 adyacente a_5UTR 66148 rs2285914 7 10714209 2 two-hit or HapMap 4697 NDUFA4 NM_002489.2 adyacente a_3UTR 225131 rs2288034 16 77803457 2 wo-hit or HapMap 729251 LOC729251 KM_001133651.1 adyacente a_3UTR 673 rs2332631 14 25562376 2 wo-hit or HapMap 1857 MOVA1 MMJW6489.2 adyacente a_3UTR 422553 rs2333660 L4 , 29464817 2 t wo-hit or HapMap 5587 3RKD1 MM_002742.1 ntron 1389 SNP Cr Coord CV BV Gen Nombre Gen Número de Acceso Localización LRG rs2337970 22 46405112 3 goldengate 400932 RP11-191L9.1 NM_001001693.1 5UTR [1267/870] rs2368940 11 119575980 2 two-hit or Hap ap 220323 OAF NM_178507.2 adyacente a_5UTR -10977 rs2381628 9 7508656 3 goldengate 158345 LOC158345 XR_017130.1 adyacente a_5UTR -30022 rs2419117 1 166855806 3 goldengate 6375 XCL1 NM_002995.1 adyacente a_3UTR -37867 rs2439553 6 13433258 2 two-hit or HapMap 51256 TBC1D7 NM_016495.2 intron -47 rs2470378 12 69512747 2 two-hit or HapMap 5801 PTPRR NM_002849.2 intron -59979 rs2505513 10 42953543 3 goldengate 55454 GALNACT-2 NM_018590.3 adyacente a_5UTR -397 rs2545391 5 66434867 2 two-hit or HapMap 4064 CD180 NM_005582.1 adyacente a_3UTR -79005 rs2555605 13 32731810 3 goldengate 90627 STARD13 NM_178008.1 intron -25797 rs256996 5 114943989 2 two-hit or HapMap 353376 TICAM2 NM_021649.3 3UTR [1742/155] rs2593693 11 22324877 2 two-hit or HapMap 57084 SLC17A6 NM_020346.1 intron -3390 rs2648774 1 197382785 3 goldengate 647201 LOC647201 XR_017465.1 adyacente a_5UTR -222021 rs265603 12 128701943 2 two-hit or HapMap 121256 TMEM132D NM_133448.1 intron -48365 rs2736000 8 127469169 3 goldengate 157638 FAM84B NM_174911.3 adyacente a_3UTR -164700 rs2745443 6 133063075 2 two-hit or HapMap 8876 VNN1 NMJ304666.1 intron -6061 rs2749928 e 131900472 3 goldengate 383 ARG1 NM_000045.2 adyacente a_5UTR -35586 rs2761843 13 50191038 3 goldengate 220107 DLEU7 NM_198989.1 intron -5661 rs2801640 5 65640474 2 two-hit or HapMap 401191 FU46010 NM_001001703.1 intron -9374 rs2834684 21 35234217 3 goldengate 861 RUNX1 NM_001754.3 intron -47087 rs2836392 21 38727745 3 goldengate 2078 ERG NM_182918.2 intron -10392 rs285550 12 40244561 2 two-hit or HapMap 29951 PDZRN4 NM_013377.2 intron -843 rs286002 5 55742226 2 two-hit or HapMap 441073 LOC441073 XR_016376.1 adyacente a_3UTR -93937 rs286007 5 55739933 2 two-hit or HapMap 441073 LOC441073 XR_016376.1 adyacente a_3UTR -96230 rs2869872 4 77410136 2 two-hit or HapMap 8987 STBD1 NM_003943.1 adyacente a_5UTR -36687 rs2922051 11 41356666 2 two-hit or HapMap 387761 LOC387761 XM_373495.4 adyacente a_3UTR -809203 rs294634 3 14875555 2 two-hit or HapMap 152273 FGD5 NM_152536.2 intron -5084 rs2953676 3 115765942 3 goldengate 26137 ZBTB20 NM_015642.2 intron -64014 rs2976501 8 93045739 3 goldengate 862 RUNX1T1 NM_175636.1 intron -3818 rs2986574 1 182173237 2 two-hit or HapMap 23127 GLT25D2 NM_015101.2 3UTR [1648/1280] rs2992630 12 113757258 2 two-hit or HapMap 6926 TBX3 NM_005996.3 adyacente a_5UTR -150906 rs3094694 6 30559883 3 goldengate 3133 HLA-E NM_005516.4 adyacente a_5UTR -5367 rs324454 5 55238308 2 two-hit or HapMap 133396 IL31RA NM_139017.3 intron -435 rs332185 10 28953315 3 goldengate 51322 WAC NM_100264.1 adyacente a_3UTR -3837 rs353111 2 165845275 3 goldengate 643397 LOC643397 XR_016377.1 adyacente a_5UTR -4677 rs3731714 2 201769065 3 goldengate 843 CASPIO NMJJ01230.3 intron -149 rs3731818 2 86222315 3 goldengate 55037 PTCD3 NM_017952.4 3UTR [4121/476] rs37389 5 35120937 2 two-hit or HapMap 5618 PRLR NM_000949.2 intron -439 rs3740808 11 89531857 2 two-hit or HapMap 10003 NAALAD2 NM_005467.2 intron -198 rs3752958 14 99252440 2 two-hit or HapMap 10858 CYP46A1 NM_006668.1 intron -151 rs3756616 5 179333453 2 two-hit or HapMap 55819 RNF130 NM_018434.4 intron -3341 rs3763131 5 179666918 2 two-hit or HapMap 9945 GFPT2 NM_005110.1 intron -9 rs3764333 16 83350762 2 two-hit or HapMap 9100 USP10 NM_005153.1 intron -183 rs376691 21 19278409 3 goldengate 653214 PPIAL3 XM_926499.2 adyacente a_3UTR -125703 rs3771910 2 175455713 3 goldengate 1123 CHN1 NM_001822.3 intron -4611 rs3775866 4 68126775 2 two-hit or HapMap 26228 STAP1 NM_012108.1 intron -1203 rs3777981 6 117841948 3 goldengate 6098 ROS1 NM_002944.2 intron -2166 rs3816334 2 233417050 3 goldengate 26058 TNRC15 NM_015575.2 coding [50/159] SNP Cr Coord cv ' BV Gen Nombre Gen Número de Acceso Localización LRG rs3817269 1 15928951 3 goldengate 23207 PLEKHM2 XM_290944.5 coding [72/60] rs3827494 3 46577235 3 goldengate 79442 LRRC2 NM_024512.2 intron -5436 rs383673 16 77789947 2 two-hit or HapMap 729251 LOC729251 XM_001133651.1 adyacente a_3UTR -14183 rs3848079 13 31324730 3 goldengate 122042 RXFP2 NM_130806.2 adyacente a_3UTR -49721 rs3891371 5 113862562 2 two-hit or HapMap 3781 KCNN2 NM_170775.1 adyacente a_3UTR -2466 rs3898966 12 33972394 2 two-hit or HapMap 84920 ALG10 NM_032834.2 adyacente a_5UTR -94089 rs39O3470 3 24483025 2 two-hit or HapMap 644990 LOC644990 XM_932564.2 adyacente a_5UTR -27371 rs3935670 2 235076947 3 goldengate 10123 ARL4C NM_005737.3 adyacente a_5UTR -6515 rs3936139 19 2538576 2 two-hit or HapMap 2788 GNG7 NM_052847.1 adyacente a_5LJTR -32390 rs408265 6 2787424 2 two-hit or HapMap 1992 SERPINB1 NM_030666.2 adyacente a_5UTR -344 rs4142602 13 92733503 3 goldengate 10082 GPC6 NM_005708.2 intron -55633 rs4339871 1 217499653 3 goldengate 127018 LYPLAL1 NM_138794.1 adyacente a_3UTR -47870 rs4396907 3 83989494 2 two-hit or HapMap 643665 LOC643665 XM_931729.2 adyacente a_5UTR -1099351 rs4407980 9 36942743 3 goldengate 5079 PAX5 NM_016734.1 intron -13803 rs4432612 3 43675296 2 two-hit or HapMap 51099 ABHD5 NM_016006.3 adyacente a_5UTR -32083 rs4438696 3 31900672 2 two-hit or HapMap 114884 OSBPL10 NM_017784.3 intron -4346 rs4477896 2 33805917 2 two-hit or HapMap 151325 MYADML XR_017840.1 3UTR [1090/858] rs4490160 2 38582415 2 two-hit or HapMap 220717 RPLPO-like XR_017813.1 adyacente a_3UTR -18830 rs4495512 9 29908652 3 goldengate 401497 LOC401497 XM_376822.3 adyacente a_3UTR -470281 rs4505714 3 84573028 2 two-hit or HapMap 643665 LOC643665 XM_931729.2 adyacente a_5UTR -515817 rs4658673 1 243108935 3 goldengate 3192 HNRNPU NM_004501.3 adyacente a_5UTR -14485 rs4663726 2 237925851 2 two-hit or HapMap 1293 COL6A3 NM_004369.2 intron -53 rs4666167 2 29208807 3 goldengate 79745 CLIP4 NM_024692.3 intron -192 rs4691340 4 157569429 2 two-hit or HapMap 646865 hCG_1814936 XM_933981.2 adyacente a_3UTR -205667 rs4724620 7 47757437 2 two-hit or HapMap 168507 PKD1L1 NM_138295.2 adyacente a_3UTR -23378 rs4761829 12 50367232 2 two-hit or HapMap 6334 SCN8A NM_014191.1 coding [90/38] rs4782497 16 87546780 2 two-hit or HapMap 863 CBFA2T3 NM_175931.1 intron -11671 rs4821359 22 33802273 3 goldengate 91464 ISX NM_001008494.1 intron -6238 rs485499 3 161228557 3 goldengate 730109 LOC730109 XM_001132464.1 intron -1242 rs4856541 3 84815560 3 goldengate 643665 LOC643665 XM_931729.2 adyacente a_5UTR -273285 rs4861960 4 182609327 2 two-hit or HapMap 728081 hCG_2025798 XM_001127776.1 adyacente a_5UTR -292031 rs4888984 16 78066835 2 two-hit or HapMap 4094 MAF NM_005360.3 adyacente a_3UTR -118897 rs4889197 16 71952116 2 two-hit or HapMap 388289 C16orf47 NM_207385.1 adyacente a_5UTR -216269 rs4917076 7 49678523 2 two-hit or HapMap 727813 LOC727813 XM_001125862.1 adyacente a_3UTR -8606 rs4917690 10 97224981 3 goldengate 10580 SORBS1 NM_024991.1 intron -15801 rs4968656 17 58970691 2 two-hit or HapMap 81033 KCNH6 NM_030779.2 intron -936 rs4982704 14 22433377 2 two-hit or HapMap 55147 RBM23 NM_018107.3 adyacente a_3UTR -6438 rs562966 11 119581326 2 two-hit or HapMap 220323 OAF NM_178507.2 adyacente a_5UTR -5631 rs573320 15 52465606 2 two-hit or HapMap 440279 UNC13C XM_930037.2 ntron -6932 rs5755467 22 33695091 3 goldengate 91464 SX NM_001008494.1 adyacente a_5UTR -97039 rs577562 1 67038563 2 two-hit or HapMap 10022 NSL5 NM_005478.3 ntron -755 rs588036 6 150740367 2 two-hit or HapMap 389434 YD NM_203395.1 ntron -8329 rs6014133 20 52734118 2 two-hit or HapMap 55816 DOK5 NM_018431.3 adyacente a_3UTR -33001 rs6021658 20 50105671 3 goldengate 55734 ZFP64 NM_199427.1 adyacente a_3UTR 28286 rs6021668 20 50110836 3 goldengate 55734 ZFP64 NM_199427.1 adyacente a_3UTR 23121 rs6021688 20 50126404 3 goldengate 55734 -FP64 MM_199427.1 adyacente a_3UTR 7553 rs6121904 20 50137102 3 goldengate 149986 LSM14B M_144703.1 ntron 274 SNP Cr Coord CV BV Gen Nombre Gen Número de Acceso Localización LRG rs617206 18 6975631 2 two-hit or HapMap 284217 LAMA1 NM_005559.2 coding [105/11] rs635647 9 3252803 3 goldengate 5991 RFX3 NM_002919.2 intron -132 rs6427916 1 199907444 3 goldengate 89796 NAV1 NM_020443.2 intron -22268 rs6449558 5 61121309 2 two-hit or HapMap 643307 LOC643307 XR_016336.1 adyacente a_3UTR -42219 rs6518223 21 45538977 2 two-hit or HapMap 257103 C21orf86 NM_153454.1 coding [497/0] rs6583661 10 82553590 3 goldengate 642666 LOC642666 XM_931000.2 adyacente a_3UTR -150122 rs6592852 11 78921073 2 two-hit or HapMap 646112 LOC646112 XR_017652.1 adyacente a_5UTR -455131 rs6596293 5 135544606 2 two-hit or HapMap 4090 SMAD5 NM_005903.5 3UTR [3538/1715] rs6656554 1 213954552 3 goldengate 7399 USH2A NM_206933.1 intron -13443 rs670310 11 69889222 2 two-hit or HapMap 8500 PPFIA1 NM_003626.2 intron -2980 rs6780332 3 29193263 2 two-hit or HapMap 131572 LOC131572 XRJ316841.1 adyacente a_3UTR -88380 rs6805546 3 72168842 2 two-hit or HapMap 642802 CCDC137P XR_016200.1 adyacente a_5UTR -49889 rs6821156 4 154491352 3 goldengate 84057 MND1 NM_032117.2 intron -621 rs6859143 5 103418563 2 two-hit or HapMap 83594 NUDT12 NM_031438.2 adyacente a_5UTR -492174 rs6907551 6 114199657 3 goldengate 4082 MARCKS NM_002356.4 adyacente a_5UTR -85592 rs7004457 8 87096982 3 goldengate 85481 PSKH2 NM_033126.1 adyacente a_3UTR -32825 rs7014552 8 143408330 3 goldengate 203062 TSNARE1 NM_145003.1 intron -1839 rs7020507 9 1705820 2 two-hit or HapMap 6595 SMARCA2 NM_139045.2 adyacente a_5UTR -299522 rs7023487 9 16142308 3 goldengate 203238 C9orf93 NM_173550.2 adyacente a_3UTR -180411 rs7036414 9 35953556 3 goldengate 56656 OR2S2 NM_019897.1 adyacente a_5UTR -5491 rs7121784 11 119664262 2 two-hit or HapMap 25833 POU2F3 NM_014352.1 intron -9922 rs7131721 12 59202879 2 two-hit or HapMap 390335 LOC390335 XM_001129525.1 adyacente a_3UTR -710255 rs7225575 17 42663046 2 two-hit or HapMap 4635 MYL4 NM_002476.2 adyacente a_3UTR -7002 rs7227797 18 44377669 3 goldengate 9811 KIAA0427 NM_014772.1 intron -22238 rs722827 4 174334592 2 two-hit or HapMap 51809 GALNT7 NM_017423.1 intron -7905 rs724479 1 102047645 2 two-hit or HapMap 118427 OLFM3 NM_058170.1 intron -3319 rs7251154 19 46997781 2 two-hit or HapMap 1084 CEACAM3 NM_001815.1 intron -4061 rs7254214 19 47060578 2 two-hit or HapMap 6223 RPS19 NM_001022.3 intron -3457 rs729829 1 9081584 3 goldengate 80045 GPR157 NMJ324980.4 adyacente a_3UTR -5479 rs7299117 12 126267913 2 two-hit or HapMap 121296 LOC121296 XR_016630.1 adyacente a_3UTR -340640 rs7318830 13 81035524 3 goldengate 647313 LOC647313 XM_934711.2 adyacente a_3UTR -719804 rs7325070 13 83079295 3 goldengate 114798 SLITRK1 NM_052910.1 adyacente a_3UTR -270049 rs733182 22 41984895 3 goldengate 80274 SCUBE1 NM_173050.1 intron -610 rs7465573 8 61311677 3 goldengate 767 CA8 NM_004056.4 intron -4185 rs7515996 1 192591449 2 two-hit or HapMap 647167 LOC647167 XR_017690.1 adyacente a_3UTR -165354 rs7554157 1 191755792 3 goldengate 79577 CDC73 NMJJ24529.3 adyacente a_3UTR -268113 rs7560961 2 35803292 3 goldengate 51232 CRIM1 NM_016441.1 adyacente a_5UTR -633609 rs756634 22 21728638 3 goldengate 27156 RTDR1 NM_014433.2 adyacente a_3UTR -2955 rs763373 11 12796918 2 two-hit or HapMap 7003 TEAD1 NM_021961.3 intron -43455 rs7649851 3 6255410 2 two-hit or HapMap 2917 GRM7 NM_181875.1 adyacente a_5UTR -622517 rs7650598 3 172622161 3 goldengate 23043 TNIK NM_015028.1 intron -38329 rs7701284 5 145908423 2 two-hit or HapMap 134391 GPR151 NM_194251.2 adyacente a_5UTR -32554 rs7737236 5 116663689 2 two-hit or HapMap 728342 LOC728342 XM_001129097.1 adyacente a_5UTR -115415 rs7747934 6 156521972 3 goldengate 57492 ARID1B NM_020732.2 adyacente a_5UTR -618806 rs7801303 7 40884595 2 two-hit or HapMap 79783 C7orfl0 NM_024728.1 adyacente a_3UTR -17713 rs7842021 8 23135149 3 goldengate 8797 TNFRSF10A NM_003844.2 intron -3065 rs7897163 10 18454226 2 two-hit or HapMap 729183 LOC729183 XM_001129594.1 adyacente a_5UTR -14559 SNP Cr Coord 1 BV Gen Nombre Gen Número de Acceso Localización LRG rs7930843 1] 109456617 2 two-hit or Hap ap 728579 LOC728579 XM_001127833.1 adyacente a_3UTR -11784 rs7955850 12 6045840 2 two-hit or HapMap 7450 VWF NM_000552.3 intron -1140 rs7970928 12 69902492 2 two-hit or HapMap 7103 TSPAN8 NM_004616.2 adyacente a_5UTR -64446 rs798332 7 77746864 3 goldengate 9863 MAGI2 NM_012301.3 intron -23030 rs7989372 13 40819933 3 goldengate 79612 NARG1L NM_018527.2 intron -7347 rs7999382 13 96739498 3 goldengate 10150 MBNL2 NM_144778.2 intron -12834 rs8006322 14 78724765 2 two-hit or HapMap 9369 NRXN3 NM_138970.2 intron -90670 rs8015746 14 51028062 2 two-hit or HapMap 729335 LOC729335 XM_001133397.1 adyacente a_5UTR -94459 rs8049155 16 52912715 2 two-hit or HapMap 728792 LOC728792 XM_001128492.1 intron -1776 rs8051512 16 83338602 2 two-hit or HapMap 9100 USP10 NM_005153.1 intron -1822 rs8083791 18 63596654 3 goldengate 92126 DSEL NMJD32160.2 adyacente a_5UTR -261707 rs809193 6 112127107 3 goldengate 2534 FYN NM_153047.1 intron -304 rs8109493 19 14464932 2 two-hit or HapMap 10755 GIPC1 NM_202467.1 intron -208 rs837511 12 123613242 2 two-hit or HapMap 9612 NCOR2 NM_006312.2 adyacente a_5UTR -44449 rs842796 1 191169038 3 goldengate 730190 LOC730190 XM_001132852.1 adyacente a_3UTR -61180 rs847126 2 176645080 3 goldengate 344191 EVX2 XM_292968.6 adyacente a_3UTR -8001 rs869026 5 138604699 2 two-hit or HapMap 9782 MATR3 NM_199189.1 adyacente a_5UTR -32992 rs878472 13 81069137 3 goldengate 647313 LOC647313 XM_934711.2 adyacente a_3UTR -686191 rs879772 3 193928757 3 goldengate 2257 FGF12 NM_004113.3 adyacente a_5UTR -691 rs882169 11 61448706 3 goldengate 728718 LOC728718 XM_001128272.1 adyacente a_5UTR -3659 rs886090 9 135189324 3 goldengate 6838 SURF6 NM_006753.3 coding [119/93] rs9036 3 14505725 3 goldengate 6533 SLC6A6 NM_003043.2 adyacente a_3UTR -1751 rs910545 22 26276986 3 goldengate 4330 MN1 NM_002430.2 adyacente a_3UTR -197279 rs911064 20 61370750 3 goldengate 55738 ARFGAP1 NMJ.75609.1 adyacente a_5UTR -3860 rs9322114 6 148333734 2 two-hit or HapMap 23328 SASH1 NM_015278.3 adyacente a_5UTR -371688 rs936056 17 71099379 2 two-hit or HapMap 728272 -99 XM_001127042.1 coding [87/9] rs938372 2 88780884 3 goldengate 22934 RPIA NM_144563.2 intron -187 rs9390613 6 149166809 3 goldengate 10090 UST NM_005715.1 intron -56303 rs952656 8 124196776 3 goldengate 93594 WDR67 NM_145647.1 intron -4700 rs9568036 13 47869937 3 goldengate 10161 P2RY5 NM_005767.3 adyacente a_3UTR -13338 rs9651511 10 125546364 2 two-hit or HapMap 119587 CPXM2 NMJ.98148.1 intron -1118 rs9788864 16 22921056 2 two-hit or HapMap 57478 USP31 NM_020718.2 adyacente a_3UTR -65812 rs9818995 3 84306747 2 two-hit or HapMap 643665 LOC643665 XM_931729.2 adyacente a_5UTR -782098 rs9822040 3 127410451 3 goldengate 10840 ALDH1L1 NM_012190.2 adyacente a_5UTR -28276 rs9842818 3 147550430 2 two-hit or HapMap 440981 LOC440981 XM_496663.3 adyacente a_3UTR -41407 rs9855978 3 80171732 3 goldengate 6091 ROBOl NM_002941.2 adyacente a_5UTR -449981 rs9869315 3 177037998 3 goldengate 254827 NAALADL2 NM_207015.1 adyacente a_3UTR -31876 rs9978582 21 46312815 3 goldengate 1292 COL6A2 NM_001849.2 adyacente a_5UTR -29655 rs885782 6 161909893 2 two-hit or HapMap 5071 PARK2 NM_004562.1 ntron -484 rs2723264 12 38938787 2 two-hit or HapMap 120892 LRRK2 NM_198578.2 ntron -762 rs2022991 5 162168159 3 goldengate 5071 PARK2 NM_013988.1 ntron -41229 rs4709517 161759771 2 two-hit or HapMap 5071 PARK2 NM_004562.1 ntron -31872 rs9347686 163091960 2 two-hit or HapMap 135138 PACRG NM_152410.1 ntron 22547 rs3016563 5 162112846 2 two-hit or HapMap 5071 PARK2 NM_013988.1 ntron 13948 rs3733449 ' J 30791345 2 wo-hit or HapMap 5622 5NCA NM_000345.2 adyacente a_3UTR 74383 rs94S6734 í 5 L62394883 goldengate 5071 3ARK2 NM_004S62.1 ntron 230 rsl893098 f L62196867 goldengate 5071 3ARK2 MM_013988.1 ntron 69937 SNP Cr Coord CV BV Gen Nombre Gen Número de Acceso Localización LRG rs6926642 6 162190971 3 goldengate 5071 PARK2 NM_013988.1 intron -64041 rs6942109 6 161706684 3 goldengate 5071 PARK2 NM_013988.1 intron -5457 rs7298930 12 39005767 2 two-hit or HapMap 120892 LRRK2 NM_198578.2 intron -2392 rsll964284 6 161732616 2 two-hit or HapMap 5071 PARK2 NM_004562.1 intron -4717 rsll564173 12 39036738 2 two-hit or HapMap 120892 LRRK2 NM_198578.2 intron -482 rs9347654 6 162840827 2 two-hit or HapMap 5071 PARK2 NM_004562.1 adyacente a_5UTR -56332 rs6906519 6 161811378 3 goldengate 5071 PARK2 NM_013988.1 intron -78498 rsl0878246 12 38918366 2 two-hit or HapMap 120892 LRRK2 NM_198578.2 intron -194 rslll75655 12 38909994 2 two-hit or HapMap 120892 LRRK2 NM_198578.2 intron -2349 rsll564203 12 39010848 2 two-hit or HapMap 120892 LRRK2 NM_198578.2 intron -2319 rsll829088 12 39014046 2 two-hit or HapMap 120892 LRRK2 NM_198578.2 intron -990 rsl0878307 12 38958256 2 two-hit or HapMap 120892 LRRK2 NM_198578.2 coding [96/74] rsl2525605 6 163628328 3 goldengate 135138 PACRG NM_152410.1 adyacente a_5UTR -25473 rsl907632 12 38936769 2 two-hit or HapMap 120892 LRRK2 NM_198578.2 intron -548 rs2000594 6 162161095 3 goldengate 5071 PARK2 NM_013988.1 intron -34165 rsl0857057 4 119182533 3 goldengate 9348 NDST3 NM_004784.1 intron -7338 rslll34974 5 175714565 2 two-hit or HapMap 57179 KIAA1191 NM_020444.2 intron -615 rsl2589063 14 88507193 2 two-hit or HapMap 123016 TTC8 NM_198310.2 adyacente a_3UTR -93105 rsl329579 9 81749779 3 goldengate 347119 LOC347119 XM_928399.1 adyacente a_3UTR -79448 rs281357 17 19683106 2 two-hit or HapMap 9706 ULK2 NM_014683.2 intron -618 rs2862909 13 23999167 3 goldengate 143 PARP4 NM_006437.2 adyacente a_5UTR -14219 rs3764286 16 83298844 2 two-hit or HapMap 9100 USP10 NM_005153.1 intron -7626 rs4350947 3 123227539 3 goldengate 286676 ILDR1 NM_175924.2 adyacente a_5UTR -3819 rs7146193 14 88516549 2 two-hit or HapMap 123016 TTC8 NM_198310.2 adyacente a_3UTR -102461 rs7218605 17 74323398 2 two-hit or HapMap 57602 USP36 NM_025090.2 intron -1169 rs7539016 1 231556122 2 two-hit or HapMap 84451 KIAA1804 NM_032435.1 intron -54 rs761267 20 41896884 2 two-hit or HapMap 728240 LOC728240 XM_001126959.1 adyacente a_5UTR -79534 rs7653784 3 14848798 2 two-hit or HapMap 152273 FGD5 NM_152536.2 intron -10691 rs7924316 11 2130023 2 two-hit or HapMap 51214 IGF2AS NM_016412.1 adyacente a_3UTR -3553 rs9382517 6 55586555 3 goldengate 54511 HMGCLL1 NM_019036.1 adyacente a_5UTR -34611 rs5760103 22 22578781 2 two-hit or HapMap 4282 MIF NM_002415.1 adyacente a_3UTR -11372 rs4865845 5 53913691 2 two-hit or HapMap 112574 SNAG1 NM_052870.1 adyacente a_3UTR -62265 rs7667015 4 42634138 2 two-hit or HapMap 389207 LOC389207 XM_371694.4 intron -25528 rs7830101 8 39527585 3 goldengate 1587 ADAM3A NR_001569.1 adyacente a_5UTR -28061 rs4583999 6 156527609 2 two-hit or HapMap 57492 ARID1B NM_020732.2 adyacente a_5UTR -613169 rs741365 12 5554582 2 two-hit or HapMap 57101 TMEM16B NM_020373.1 intron -679 rsll235935 11 69856226 2 two-hit or HapMap 8500 PPFIA1 NM_003626.2 intron -378 rsl740377 6 112149811 2 two-hit or HapMap 2534 FYN NM_153048.1 intron -1853 rsll070178 15 37329814 2 two-hit or HapMap 400360 C15orf54 NM_207445.1 adyacente a_5UTR -397 rs886016 9 135545450 2 two-hit or HapMap 1757 SARDH NM_007101.2 coding [127/20] rs4813430 20 21484958 3 goldengate 4821 NKX2-2 NM_002509.2 adyacente a_5UTR -42294 rs3128501 9 89518960 3 goldengate 1612 DAPK1 NMJD04938.2 adyacente a_3UTR -5591 rs7105372 11 8700795 2 two-hit or HapMap 6764 ST5 NM_213618.1 intron -3401 rs6508999 19 47014387 2 two-hit or HapMap 1084 CEACAM3 NM_001815.1 adyacente a_3UTR -6958 rsl532763 11 121010533 2 two-hit or HapMap 6653 SORL1 NM_003105.3 adyacente a_3UTR -4912 rsl039189 18 49731452 2 two-hit or HapMap 8932 MBD2 NM_003927.3 adyacente a_3UTR -203121 rs6047472 20 21478788 2 two-hit or HapMap 4821 NKX2-2 NM_002509.2 adyacente a_5UTR -36124 SNP Cr Coord CV BV Gen Nombre Gen Número de Acceso Localización LRG rs6592941 7 51642120 2 two-hit or HapMap 642663 LOC642663 XM_926118.1 adyacente a_3UTR -224163 rs7027515 9 126213030 3 goldengate 5695 PSMB7 NM_002799.2 intron -1422 rs4267850 5 77683520 2 two-hit or HapMap 727723 LOC727723 XM_001125292.1 adyacente a_5UTR -6313 rs4072198 3 62187107 2 two-hit or HapMap 5793 PTP G NM_002841.2 intron -4832 rsll753244 6 165013078 2 two-hit or HapMap 728275 LOC728275 XM_001127046.1 adyacente a_3UTR -334296 rsll56143 7 136740077 2 two-hit or HapMap 9162 DGKI NM_004717.2 intron -3084 rs2399095 3 107605293 2 two-hit or HapMap 728784 LOC728784 XM_001128470.1 adyacente a_5UTR -19073 rs4285366 6 8839136 3 go!dengate 389365 LOC389365 XM_371797.3 adyacente a_5UTR -147723 rs902074 5 163173200 2 two-hit or HapMap 391844 LOC391844 XR_016645.1 adyacente a_5UTR -45558 rsl634079 4 150007154 2 two-hit or HapMap 4306 NR3C2 NM_000901.1 adyacente a_5UTR -424181 rsl0153012 15 92684286 2 two-hit or HapMap 55784 MCTP2 NM_018349.2 intron -146 rsl934115 9 23093266 3 goldengate 402360 LOC402360 XR_017263.1 adyacente a_5UTR -578629 rsl5S9854 11 83741948 2 two-hit or HapMap 1740 DLG2 NM_001364.2 intron -101966 rsl0497933 2 211849925 2 two-hit or HapMap 2066 ERBB4 NM_005235.1 adyacente a_3UTR -105135 rs4433214 9 120542512 3 goldengate 442434 LOC442434 XR_016936.1 adyacente a_5UTR -99 rsl0244108 7 55119831 3 goldengate 1956 EGFR NM_201282.1 intron -57642 rs7023309 9 137587676 3 goldengate 654089 LOC654089 XM_939055.1 adyacente a_5UTR -3107 rsl0465957 1 155632002 3 goldengate 729853 -99 XM_001134256.1 adyacente a_3UTR -117789 rs2056777 3 25515395 3 goldengate 5915 RARB NM_016152.2 intron -2282 rsll927332 3 70689611 3 goldengate 401072 -99 NM_001013679.1 adyacente a_3UTR -55828 rsll071896 15 64608304 2 two-hit or HapMap 55055 ZWILCH NM_017975.2 coding [60/45]  SNP Cr Coord CV BV Gen Name Gen Access Number Location LRG rsl003167 6 43963001 3 goldengate 7422 VEGFA NM_001033756.1 adjacent to_3UTR -100799 rsl0046663 8 2983419 3 goldengate 64478 CSMD1 NM_033225.3 intron -3975 rsl0O5306 6pp631pp 631pp 631pp 631pp 631pp 631pp 631pp 631pp 631pp 631pp 631pp 631pp 631pp 6155 CAST NM_173060.1 intron -4801 rsl0078721 5 92446445 2 two-hit or HapMap 391811 LOC391811 XR_016127.1 adjacent to_5UTR -182298 rsl008577 19 46879134 2 two-hit or HapMap 1087 CEACAM7 NM_006890.10094000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000003 MM4003163 MM4003163 MM4003163 MM4003163 MM4003163 .3 intron -4795 rsl0096279 8 3212285 2 two-hit or HapMap 64478 CSMD1 NM_033225.3 intron -109 rsl0121215 9 26159347 3 goldengate 441390 -99 NM_001004352.1 adjacent to_3UTR -50941 rsl0155062 2 7184805297_281125297 7284127 7324M 7124125297 7284287 2 7284287 2 7284127 7274285 2 7284 7284 7284 7284 2 7284 2 7284 2 7284 7284 2 7284 7284 2 7284 2 7284 2 7284 2 7287 1 adjacent to_3UTR -55088 rsl0201616 2 75529368 3 goldengate 84141 TMEM166 NM_032181.1 adjacent to_3UTR -43584 rsl022242 12 69492622 2 two-hit or HapMap 5801 PTP NM_002849.2 intron -47797 rsl035316 2 golden-4g7333 ate 79843 FAM124B NM_024785.2 adjacent to_3UTR -103262 rsl0496075 2 57762864 2 two-hit or HapMap 7444 VR 2 NM_006296.3 adjacent to_5UTR -364360 rsl0511001 3 70715884 2 two-hit or HapMap 4010725999U9_8095806996996996996996996996996996996996996996996996996996999 20277623 2 two-hit or HapMap 4300 MLLT3 NM_004529.1 adjacent to_3UTR -57345 rsl0512174 9 88076394 3 goldengate 81689 ISCA1 NM_030940.3 intron -348 rslOS 16970 4 96529259 2 two-hit or HapMap 86336454 9453455 3 goldengate 3371 TNC NM_002160.1 intron -391 rsl0747874 12 59144947 2 two-hit or HapMap 390335 LOC390335 XM_0O1129525.1 adjacent to_3UTR -652323 rsl0750861 11 56937983 2 two-hit or HapMap. 1441 CSF3R NM_172313.1 adjacent to_5UTR -66302 rsl0797007 1 156562253 3 goldengate 910 CD1B NM_001764.1 adjacent to_3UTR -2150 rsl0801705 1 89272970 3 goldengate 2635 GBP3 NM_018284.1 adjacent to_5U5 882 I 64 863 -155 364 METTL4 NM_022840.2 adjacent a_3UTR rsl0916407 1 227216444 677790 3 goldengate TMEM78 NM_001040079.1 adjacent a_5UTR rsl0935844 -235562 116931 3 152 611 585 3 goldengate MED12L NM_053002.3 intron 6 169193728 -171 rsl0945364 3 goldengate 7058 THBS2 NM_003247.2 adjacent a_3UTR - 164072 rsll008264 10 31129838 3 goldengate 220929 ZNF438 NM_182755.1 adjacent a_3UTR -43731 rsll06145 3 87631095 3 goldengate 643766 LOC643766 XR_016476.1 adjacent a_3UTR -131046 rsll081496 18 9716631 226968926926926926926926926926926926926926926926926926926926926926926926726 2BR926726 226968926926926926926926926926926926926926926926926926926926926926926926926926926926926926926926926926926926726 two-time 2 91672626 226926726 226926726 226926926726 two-way number two-hit or HapMap 2114 ETS2 NMJ305239.4 intron 1 206 813 021 3 -359 rsllll9097 goldengate NM_025179.2 5362 PLXNA2 adjacent a_5UTR 10 -329 282 115 684 622 2 rslll96543 two-hit or HapMap NHLRC2 NM_198514.2 374354 -26180 adjacent a_3UTR rsl 5118252 14305 12 3 goldengate 3741 KCNA5 NM_002234.2 adjacent to_3UTR -92042 rsll50996 12 31960144 2 two-hit or HapMap 55196 C12orf35 NM_018169.2 adjacent to_5UTR -43476 rsll564252 12 3 9100000 2 two-hit or HapMap 283463 MUC19 XM_497341.3 ntron • 276  SNP Cr Coord CV BV Gen Name Gen Access Number Location LRG rsll655589 17 58961895 2 two-hit or HapMap 81033 KCNH6 NM_173092.1 intron -260 rsll674954 2 1262038 3 goldengate 54221 SNTG2 NM_018968.2 adjacent to_3UTR -8114 2 860194 8gate18194 8gate18694 goldengate ST3GAL5 NM_003896.2 adjacent a_5UTR -49306 rsll694263 2 237 855 128 2 two-hit or HapMap LOC728245 728245 -14194 XM_001126970.1 adjacent a_5UTR rsll715563 3 LOC643665 643665 83873910 3 goldengate XM_931729.2 adjacent a_5UTR -1214935 114525871 2 rsll738500 5 two-hit or HapMap 55521 TRIM36 NM_018700.3 intron -1243 rsll860440 16 1697619 2 two-hit or HapMap 23162 MAPK8IP3 NM_001040439.1 intron -960 rsll893869 2 106032330 3 goldengate 84417 C2orf40 NM_032411.1 adjacent to_5UTR -162156796 7320 rsl2081567 1 151467611 3 goldengate 391102 LOC391102 XR_016996.1 adjacent to_3UTR -2505 rsl2119878 1 9141308 2 two-hit or HapMap 727721 LOC727721 XM_001123441.1 adjacent to_3UT R -17730 rsl2144940 1 99705926 3 goldengate 9890 LPPR4 NM_014839.3 adjacent to_3UTR -158198 rsl2353255 9 20911332 3 goldengate 54914 KIAA1797 NM_017794.2 intron -2327 rsl2412945 10 54468113 2U-4676162U 2A-4676162U 2A-4676162U 2AA-4AJJJ-1AAAAAA 2 two-hit or HapMap 56659 KCNK13 NM_022054.2 intron -40375 rsl2495688 3 147525535 3 goldengate 440981 LOC440981 XM_496663.3 adjacent to_3UTR -66302 rsl2520264 5 7698474 2 two-hit or HapM519506105105105_5105105105105_5105105105_5105105105105105105105105105105105105105105105105105105105105106106106106106106106106106105105105105105105105105106106106105106106106106106106106105 -hit or HapMap 2230 FDX1 NM_004109.3 adjacent to_3UTR -28695 rsl2672177 7 101813787 3 goldengate 79706 PR RIP1 NM_024653.1 adjacent to_5UTR -10028 rsl267658 15 81011209 2 two-hit or HapMap599296299299296299299299299296299296299296299296299296299296299296299296296299296299296299296299296299296299296296909296296296299296296299296296296296296909296296299 -hit or HapMap 1012 CDH13 NM_001257.3 intron -22512 rsl2976749 19 43811334 2 two-hit or HapMap 27335 EIF3K NM_013234.2 intron -2752 rsl2987286 2 148380038 3 goldengate 92 ACVR2A NM_001616.3 intron -610801.3 intron -610801.3 intron -610801.3 intron -610801.3 intron -610801.3 intrl -610801.33 9 81776189 3 goldengate 347119 LOC347119 XM_928399.1 adjacent to_3UTR -105858 rsl364658 2 148324278 3 goldengate 92 ACVR2A NM_001616.3 intron -5032 rsl365461 18 34356804 3 goldengate 388474 LOC388473 2R_372165-1R7372165-1R7371-2R7372165-1R7372165-1RJ2163-JR5372162-1RJ2162-JR-372163-JRJ-1RJ-1JP-1 ADAMTS18 NM_199355.1 intron -156 rsl370699 2 86025785 3 goldengate 8869 ST3GAL5 NM_003896.2 adjacent to_5UTR -56137 rsl385331 3 197835612 3 goldengate 375387 LRRC33 NM_198565.1 adjacent to_5UTR -15910091009 284_189p1309 2p1309 2p1309 2p1309 2p1309 2p1309 2p1309 2p1309 2p1303 2p1309 2p1309 2p1304183p10004 285_149p1303 2p1309181p3164p1p3151p1303 2p1304_189p1304 2p1303_149p1304 2p1303 2p1304 2p1309 2p1304 2p1309 2p1309 2p1309 2p1303 2p1309 2p1309 2p1 [390/593] rsl447665 3 127444361 3 goldengate 644662 LOC644662 XM_933903.2 adjacent to_5UTR -23968 rsl480691 8 14808735 2 two-hit or HapMap 137868 SGCZ NM_139167.2 intron -330730 rsl4999007_28928976_00928976_00928976_700928976_7009_28976_007_28976_007_28976-7009_28976_007_28976_7006_976_76-009_28976_7-009_06_006_286_976_07_006_28976_7_006_28976_7_006_28976_7_006_28976_07_006_286_976_07_006_289_06_976_7-976_07_009_06_976_7-006 rsl50858 17 3479419 2 two-hit or HapMap 23729 CARKL NM_013276.2 intron -829 rsl511241 11 78746402 2 two-hit or HapMap 729746 LOC729746 XM_00U31186.1 adjacent to_5UTR -2 81322 rsl521937 2 53515720 3 goldengate 51130 ASB3 NM_145863.1 adjacent to_3UTR -234902 rsl527101 12 77063452 2 two-hit or HapMap 89795 NAV3 NM_014903.3 intron -828 rsl537685 10 116894920 3 golden1388303 3301498 3301 8493 3301 8493 3301 8493 3301 8493 3301 154215 NKAIN2 NM_001040214.1 intron -131696 rsl573465 21 34871068 3 goldengate 1827 RCAN1 NM_203417.1 intron -37379 rsl62216 3 7833048 2 two-hit or HapMap 2917 GRM7 NM_000844.2 adjacent to_3UTR -7709297297299299297299299299297299299299297297297299299297297297299299297299297297297299299299299297299299297299299929929929992972999297299929729992972999119119119119119119119119119119119119119119119119119119119119119119111 a_3UTR -189426 rsl675247 18 3608506 3 goldengate 649446 FU35776 NM_001039796.1 adjacent to_3UTR -20156 rsl708694 18 32321976 3 goldengate 80206 FHOD3 NMJ325135.2 intron -13917 rsl725265 1 23801-1 2 two-hit or HapMap 645641 LOC645641 XM_928656.1 adjacent to_5UTR -466370  SNP Cr Coord CV 1 BV Gen Name Gen Access Number Location LRG rsl7740213 8 24430274 2 two-hit or HapMap 8756 ADAM7 NM_003817.1 adjacent a_3UTR -8103 rsl78214 14 26015206 2 two-hit or HapMap 4857 NOVA1 NM_006491.2 intron -792351.2l -796351.2 intron 11 92040229 2 two-hit or HapMap 120114 FAT3 XM_926199.2 intron -29969 rsl828034 4 148309936 3 goldengate 83894 TTC29 NMJ331956.1 adjacent to_5UTR -223497 rsl836726 2 211892263 3 goldengate 2066 ERBB.19600697-677627697-777627697-6776276-96_76_6_6_66-6776276-96_6_6_6_6_66-6776276196006776 hit or HapMap 285888 CNPY1 XM_001129537.1 intron 6 165012190 rsl874404 -1900 3 728 275 LOC728275 goldengate XM_001127046.1 adjacent a_3UTR rsl887279 -333,408 23,127 1 182 176 783 3 goldengate GLT25D2 NM_015101.2 intron 7 138314672 2 -239 rsl888349 two-hit or HapMap 92092 NM_080660 ZC3HAV1L .2 adjacent to_3UTR -46320 rsl898111 15 45679590 2 two-hit or HapMap 80031 SEMA60 NM_020858.1 adjacent to_5UTR -118388 rsl901818 16 79761626 2 two-hit or HapMap 114780 PKD1L2 NM_052892.2 intron -423 31704 10 129229799 3 goldengate 1793 DOCK1 NM_001380.2 adjacent to_3UTR -89029 rsl940814 18 14061989 3 goldengate 162655 ZNF519 NM_145287.1 adjacent to_3UTR -32735 rsl952198 14 69409508 2 two-hit or HapM513813873881381338713838813838738813838738813838713838538813838738338338713838538833871383853883387138385388338713833873851381351381333873851381351381351383387387387387387383 goldengate GNG2 NM_053064.2 54331 -26473 adjacent a_5UTR rsl962293 9 2100744 3 goldengate 6595 SMARCA2 NM_139045.2 intron 6 159025401 -327 rsl979541 3 goldengate 94120 SYTL3 NM_001009991.1 rs2011207 3 104449957 -1392 intron 3 adjacent goldengate 644681 LOC644681 XM_927781.2 a_3UTR -290967 rs2026690 13 46381178 2 two-hit or HapMap HTR2A NM_000621.2 3356 -13002 adjacent a_5UTR rs2041611 18 14077956 3 goldengate NM_145287.1 162655 ZNF519 rs2047214 4 -16768 adjacent a_3UTR goldengate 11150840 3 9957 -111 205 HS3ST1 NM_005114.2 adjacent a_5UTR rs2053380 7 12367362 2 two-hit or HapMap 221806 FU14712 XM_371878.5 coding [64/40] rs205752S 13 92755817 3 goldengate 10082 GPC6 NM_005708.2 intron -77947 rs2063239 12 817983 72 3 goldengate 160335 TMTC2 NM_152588.1 intron -15356 rs2070862 17 1595044 2 two-hit or HapMap 5345 SERPINF2 NM_000934.1 coding [4/58] rs207620 13 32011403 3 goldengate 10443 RP11-298P3.3 NM_014887.1 adjacent_ a5 1 23650949 3 goldengate 55616 DDEFL1 NM_017707.2 intron -341 rs2113447 18 51827201 3 goldengate 642484 FU45743 NM_001039759.1 intron -5068 rs2129530 11 11354861 2 two-hit or HapMap 374378 GALNTL4 6164166164166164166164166164166164166164166164166164166164166164166164166164166164166164166164166164166164166164166164166164166164166164166164166164166164166164166166164166166166161 .2 intron -669 rs2163307 8 4293028 2 two-hit or HapMap 64478 CSMD1 NM_033225.3 intron -28033 rs218280 4 55119046 3 goldengate 3815 KIT NM_000222.1 adjacent to_5UTR -99872 rs2219925 18 47730531 NM_1600_D340_3333_33 adjacent_ NM3300_D1 to NM331 rs2220971 13 96826076 2 two-hit or HapMap 10150 MBNL2 NM_144778.2 intron -9268 rs222717 14 23161964 2 two-hit or HapMap 10202 DHRS2 NM_005794.2 adjacent to_5UTR -13458 rs2233568 9 35947355199S2 NM2149991097 9921077 9N2192107 992167 code9 ] rs2235490 16 1683512 2 two-hit or HapMap 90861 HN1L NM_144570.2 intron -1525 rs2235616 20 48002534 2 two-hit or HapMap 55905 ZNF313 NM_018683.3 3UTR [448/1295] rs2241780 3 14495168M 2ap 33336006 2ap-NM3493 .2 ntron -88 rs2242330 4 58129844 2 two-hit or HapMap 26228 STAP1 NM_012108.1 ntron -60 rs2242383 12 75962738 2 two-hit or HapMap 144455 E2F7 NM_203394.1 ntron -32 rs2253729 7 139589358 2 two-hit8 Ha1M NM_030647.1 adjacent to_5UTR 66148 rs2285914 7 10714209 2 two-hit or HapMap 4697 NDUFA4 NM_002489.2 adjacent to_3UTR 225131 rs2288034 16 77803457 2 wo-hit or HapMap 729251 LOC729251 KM_0063331 MOVA1 MMJW6489.2 adjacent to_3UTR 422553 rs2333660 L4, 29464817 2 t wo-hit or HapMap 55873RKD1 MM_002742.1 ntron 1389  SNP Cr Coord CV BV Gen Name Gen Access Number Location LRG rs2337970 22 46405112 3 goldengate 400932 RP11-191L9.1 NM_001001693.1 5UTR [1267/870] rs2368940 11 119575980 2 two-hit or Hap ap 220323 OAF NM_178507.2 adjacent to_5U -10977 rs2381628 9 7508656 3 goldengate 158345 LOC158345 XR_017130.1 adjacent to_5UTR -30022 rs2419117 1 166855806 3 goldengate 6375 XCL1 NM_002995.1 adjacent to_3UTR -37867 rs2439553 6 13433258 2 two-strike 2 two-hit or HapMap 5801 PTPRR NM_002849.2 intron -59979 rs2505513 10 42953543 3 goldengate 55454 GALNACT-2 NM_018590.3 adjacent to_5UTR -397 rs2545391 5 66434867 2 two-hit or HapMap 4064 CD180 -5555_5555_5555_5555 32731810 3 goldengate 90627 STARD13 NM_178008.1 intron -25797 rs256996 5 114943989 2 two-hit or HapMap 353376 TICAM2 NM_021649.3 3UTR [1742/155] rs2593693 11 22324877 2 two-hit or HapMap 57084 SLC172642682669066266-0264266266-0266266-1 1973827 85 3 goldengate 647201 LOC647201 XR_017465.1 adjacent to_5UTR -222021 rs265603 12 128701943 2 two-hit or HapMap 121256 TMEM132D NM_133448.1 intron -48365 rs2736000 8 127469169 3 goldengate 157638 FAM84B NM_173303274276273306274276273307277276273453274276273307276273307276273306273453 two -473303276273453 two -473303273303 two -476303276303 two-three or HapMap 8876 VNN1 NMJ304666.1 intron -6061 rs2749928 e 131900472 3 goldengate 383 ARG1 NM_000045.2 adjacent to_5UTR -35586 rs2761843 13 50191038 3 goldengate 220107 DLEU7 NM_198989.1 intron -5664 2R1401 1001401101001101401101401001101401 1 intron -9374 rs2834684 21 35234217 3 goldengate 861 RUNX1 NM_001754.3 intron -47087 rs2836392 21 38727745 3 goldengate 2078 ERG NM_182918.2 intron -10392 rs285550 12 40244561 2 two-hit or HapMap 29951 PDZR224 556273 543263 643-043 6373 2 two-hit or HapMap 441073 LOC441073 XR_016376.1 adjacent to_3UTR -93937 rs286007 5 55739933 2 two-hit or HapMap 441073 LOC441073 XR_016376.1 adjacent to_3UTR -96230 rs2869872 4 7741013p 2 two-hit 8987 STBD1 NM_003943.1 adjacent to_5UTR -36687 rs2922051 11 41356666 2 two-hit or HapMap 387761 LOC387761 XM_373495.4 adjacent to_3UTR -809203 rs294634 3 14875555 2 two-hit or HapMap 1522731573696363369363369363369363367363369363367363363366363 6263 3127363366363 3127366363 6263 golden ZBTB20 NM_015642.2 intron -64014 rs2976501 8 93045739 3 goldengate 862 RUNX1T1 NM_175636.1 intron -3818 rs2986574 1 182173237 2 two-hit or HapMap 23127 GLT25D2 NM_015101.2 3UTR [1648/122680 2ap75 692626 2ap 69226p 2p75 TBX3 NM_005996.3 adjacent to_5UTR -150906 rs3094694 6 30559883 3 goldengate 3133 HLA-E NM_005516.4 adjacent to_5UTR -5367 rs324454 5 55238308 2 two-hit or HapMap 133396 IL31RA NM_139017.3 intron -43533232518333312323 golden_3133323 518433332135323 golden_3133323 5185333 golden133323 5134323 golden_332318333332135323 golden_3303 golden_3303 golden_332135323 golden_3303 golden_332135323 golden_3303 golden_3323153 golden_3323153 golden_3183323 golden_3303 golden 1 adjacent to_3UTR -3837 rs353111 2 165845275 3 goldengate 643397 LOC643397 XR_016377.1 adjacent to_5UTR -4677 rs3731714 2 201769065 3 goldengate 843 CASPIO NMJJ01230.3 intron -149 rs3731818 2 86222315 3 goldenUK03 312 1/476] rs37389 5 35120937 2 two-hit or HapMap 5618 PRLR NM_000949.2 intron -439 rs3740808 11 89531857 2 two-hit or HapMap 10003 NAALAD2 NM_005467.2 intron -198 rs3752958 14 99252440 2 two-hit or HapMap461 108 NM58 or HapMap461 108 NM58 or HapMap461 108 NM58 or HapMap461 108 NM5858 .1 intron -151 rs3756616 5 179333453 2 two-hit or HapMap 55819 RNF130 NM_018434.4 intron -3341 rs3763131 5 179666918 2 two-hit or HapMap 9945 GFPT2 NM_005110.1 intron -9 rs3764333 16 833007p 2ap 91-33007p 2ap 91-33007p 2ap 91-3300710 NM_005153.1 intron -183 rs376691 21 19278409 3 goldengate 653214 PPIAL3 XM_926499.2 adjacent to_3UTR -125703 rs3771910 2 175455713 3 goldengate 1123 CHN1 NM_001822.3 intron -4611 rs3775866 4 68126775p 21-np8221101201201201201201201201201201201201202 rs3777981 6 117841948 3 goldengate 6098 ROS1 NM_002944.2 intron -2166 rs3816334 2 233417050 3 goldengate 26058 TNRC15 NM_015575.2 coding [50/159]  SNP Cr Coord cv 'BV Gen Name Gen Access Number Location LRG rs3817269 1 15928951 3 goldengate 23207 PLEKHM2 XM_290944.5 coding [72/60] rs3827494 3 46577235 3 goldengate 79442 LRRC2 NM_024512.2 intron -5436 rs389947 16 7 -8389947 16 7 -88384773 2 777-877384773 2 777 or HapMap 729251 LOC729251 XM_001133651.1 adjacent to_3UTR -14183 rs3848079 13 31324730 3 goldengate 122042 RXFP2 NM_130806.2 adjacent to_3UTR -49721 rs3891371 5 113862562 2 two-hit or HapMap 37966-39793 NM4-37966 NMR 379_7993 NM3-379_3379 ___3373 −3383 −99383 −99383 NM 9 98 9 98 9 9 9 9 9 cente 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 hit hit hit hit hit hit hit hit 24 24 24 24 24 24 hit or HapMap 84920 ALG10 NM_032834.2 adjacent to_5UTR -94089 rs39O3470 3 24483025 2 two-hit or HapMap 644990 LOC644990 XM_932564.2 adjacent to_5UTR -27371 rs3935670 2 235076947 3 goldengate 10123 AR5396 38636_6 NM-355366_6 two-a-half or HapMap 2788 GNG7 NM_052847.1 adjacent to_5LJTR -32390 rs408265 6 2787424 2 two-hit or HapMap 1992 SERPINB1 NM_030666.2 adjacent to_5UTR -344 rs4142602 13 92733503 3 goldengate 10082 GPC6 NM_00570833 -on5008336 -_555338 rs4339871 1 217499653 3 goldengate 127018 LYPLAL1 NM_138794.1 adjacent to_3UTR -47870 rs4396907 3 83989494 2 two-hit or HapMap 643665 LOC643665 XM_931729.2 adjacent to_5UTR -1099351 rs4407980 3 362761273 3RD 436 7133 3RD 436 7293 3RD 9793163 3RD31603133 3RD276136 3303163 6136136 3303163 6133 6136136 3306296 3306296 3136136 3306136 3306296 3166136 3306136 3306166 -hit or HapMap 51099 ABHD5 NM_016006.3 adjacent to_5UTR -32083 rs4438696 3 31900672 2 two-hit or HapMap 114884 OSBPL10 NM_017784.3 intron -4346 rs4477896 2 33805917 2 two-hit or HapMap 151325U890908TR [8138TR] rs4490160 2 38582415 2 two-hit or HapMap 220717 RPLPO-like XR_017813.1 adjacent to_3UTR -18830 rs4495512 9 29908652 3 goldengate 401497 LOC401497 XM_376822.3 adjacent to_3UTR -470281 rs4505714 2 8657p365_5M5365365_95_365365_95_5A5MR532364_5 -515817 rs4658673 1 243108935 3 goldengate 3192 HNRNPU NM_004501.3 adjacent to_5UTR -14485 rs4663726 2 237925851 2 two-hit or HapMap 1293 COL6A3 NM_004369.2 intron -53 rs4666167 2 29208807 CL2 golden 323 794 CL2 3 794 034 CL2 golden intron -192 rs4691340 4 157569429 2 two-hit or HapMap 646865 hCG_1814936 XM_933981.2 adjacent to_3UTR -205667 rs4724620 7 47757437 2 two-hit or HapMap 168507 PKD1L1 NM_13829TR.2 adjacent to 12Ap332328292328322322294322322292322292322324292322292322292322292 np2322ap2322p2322p2a2p233p2aaaaaaaa NM_014191.1 coding [90/38] rs4782497 16 87546780 2 two-hit or HapMap 863 CBFA2T3 NM_175931.1 intron -11671 rs4821359 22 33802273 3 goldengate 91464 ISX NM_001008494.1 intron -6238 rs485499 3 161228307 golden 7169109649644649649649649649644649649649649649649649649649649644649644649644649644649644644644644644644649647649 -1242 rs4856541 3 84815560 3 goldengate 643665 LOC643665 XM_931729.2 adjacent to_5UTR -273285 rs4861960 4 182609327 2 two-hit or HapMap 728081 hCG_2025798 XM_001127776.1 adjacent to_5UTR -29204 2MA8.300_009004 -118897 rs4889197 16 71952116 2 two-hit or HapMap 388289 C16orf47 NM_207385.1 adjacent to_5UTR -216269 rs4917076 7 49678523 2 two-hit or HapMap 727813 LOC727813 XM_001125862.1 adjacent to_3902176 981 3 goldengate 10580 SORBS1 NM_024991.1 intron -15801 rs4968656 17 58970691 2 two-hit or HapMap 81033 KCNH6 NM_030779.2 intron -936 rs4982704 14 22433377 2 two-hit or HapMap 55147 RBM23.3266266269266266269266269266266269266266 two-hit or HapMap 220323 OAF NM_178507.2 adjacent to_5UTR -5631 rs573320 15 52465606 2 two-hit or HapMap 440279 UNC13C XM_930037.2 ntron -6932 rs5755467 22 33695091 3 goldengate 91464 SX NM_00579396U5706395 -hit or HapMap 10022 NSL5 NM_005478.3 ntron -755 rs588036 6 150740367 2 two-hit or HapMap 389434 YD NM_203395.1 ntron -8329 rs6014133 20 52734118 2 two-hit or HapMap 55816 DOK5 NM_018431.3 201161106166106166106601601601601601601601601601601601601601601601601601601601601601601601606601601601601601601606601606161106161106161106161106161106161106161106161106161106161101 3 goldengate 55734 ZFP64 NM_199427.1 adjacent to_3UTR 28286 rs6021668 20 50110836 3 goldengate 55734 ZFP64 NM_199427.1 adjacent to_3UTR 23121 rs6021688 20 50126404 3 goldengate 55734 -FP64 MM_199427.1 adjacent to_3136104 M14B M_144703.1 ntron 274  SNP Cr Coord CV BV Gen Name Gen Access Number Location LRG rs617206 18 6975631 2 two-hit or HapMap 284217 LAMA1 NM_005559.2 coding [105/11] rs635647 9 3252803 3 goldengate 5991 RFX3 NM_002919.2 intron -132 rs6427916 1 199907444 3 goldengate 89796 NAV1 NM_020443.2 intron -22268 rs6449558 5 61121309 2 two-hit or HapMap 643307 LOC643307 XR_093_6162-6193_6162-6183364-6193-6163-6196_6166-6196_6163-6196_6166-6196_6166-6196-6166366R02-6193166 two-way two-times-two hit or HapMap 257103 C21orf86 NM_153454.1 coding [497/0] rs6583661 10 82553590 3 goldengate 642666 LOC642666 XM_931000.2 adjacent to_3UTR -150122 rs6592852 11 78921073 2 two-hit or HapMap 646112 LOC06165135135135135135135135135135135135135135135135135135135135135135135135135135135135135135135135135135135135135135135135135135135135135135136135135135135135135131 two-hit or HapMap 4090 SMAD5 NM_005903.5 3UTR [3538/1715] rs6656554 1 213954552 3 goldengate 7399 USH2A NM_206933.1 intron -13443 rs670310 11 69889222 2 two-hit or HapMap 8500 PPFIA1 NM_003626 2 293323323323323323263323323323263323323323263 3323323263 3323323263 296323323263 3323323293 two-hit or HapMap 131572 LOC131572 XRJ316841.1 adjacent to_3UTR -88380 rs6805546 3 72168842 2 two-hit or HapMap 642802 CCDC137P XR_016200.1 adjacent to_5UTR -49889 rs6821156 4 1544913511 MN1 621-341 18563 2 two-hit or HapMap 83594 NUDT12 NM_031438.2 adjacent to_5UTR -492174 rs6907551 6 114199657 3 goldengate 4082 MARCKS NM_002356.4 adjacent to_5UTR -85592 rs7004457 8 87096982 3 goldengate 85481306326326326326306326326 TSNARE1 NM_145003.1 intron -1839 rs7020507 9 1705820 two two-hit or HapMap 6595 SMARCA2 NM_139045.2 adjacent a_5UTR -299522 rs7023487 9 16,142,308 C9orf93 203238 NM_173550.2 3 goldengate adjacent a_3UTR -180411 rs7036414 9 35953556 3 goldengate 56656 OR2S2 adjacent NM_019897.1 a_5UTR -5491 rs7121784 11 119664262 2 two-hit or HapMap 25833 POU2F3 NM_014352.1 intron -9922 rs7131721 12 59202879 2 two-hit or HapMap 390335 LOC390335 XM_001129525.1 adjacent to_3UTR -710255MAP4255304255 465254PAP2254305 myr5275254p2254p2254p2254p2254p2254p2254p2254p2254p2254p2254p2254p2254p2254p2254p2254p2244254p2254p2254p2244254p2254p2254p2254p2254p2254p2254p2254p2254p2254p2254p2254p2254p2254p2254p2254p2254p2254p2254p2254p2p3p5254p2 .2 adjacent to_3UTR -7002 rs7227797 18 44377669 3 goldengate 9811 KIAA0427 NM_014772.1 intron -22238 rs722827 4 174334592 2 two-hit or HapMap 51809 GALNT7 NM_017423.1 intron -7905 rs7244 79 1 102047645 2 two-hit or HapMap 118427 OLFM3 NM_058170.1 intron -3319 rs7251154 19 46997781 2 two-hit or HapMap 1084 CEACAM3 NM_001815.1 intron -4061 rs7254214 19 47060578 2 two-hit or HapMap 6223 R22.3 R103 - 102210 3457 rs729829 1 9081584 3 goldengate 80045 GPR157 NMJ324980.4 adjacent to_3UTR -5479 rs7299117 12 126267913 2 two-hit or HapMap 121296 LOC121296 XR_016630.1 adjacent to_3UTR -340640 rs7318830 13 81035134139307139307139307137307139307139307137307137307139307137307137307139307139307139307 3 goldengate 114798 NM_052910.1 SLITRK1 adjacent a_3UTR rs733182 22 41984895 -270 049 3 goldengate SCUBE1 NM_173050.1 80274 intron -610 rs7465573 8 61311677 3 767 goldengate CA8 NM_004056.4 rs7515996 1 192591449 -4185 intron 2 two-hit or HapMap 647167 LOC647167 XR_017690. 1 adjacent to_3UTR -165354 rs7554157 1 191755792 3 goldengate 79577 CDC73 NMJJ24529.3 adjacent to_3UTR -268113 rs7560961 2 35803292 3 goldengate 51232 CRIM1 NM_016441.1 adjacent to_5UTR -633609 rs756634 227 777 28638 3 goldengate 27156 RTDR1 NM_014433.2 adjacent to_3UTR -2955 rs763373 11 12796918 2 two-hit or HapMap 7003 TEAD1 NM_021961.3 intron -43455 rs7649851 3 6255410 2 two-hit or HapMap 2917 GRM7 NM_181756162166266162162166166166162166162166166166166166166166166166166166166166166166166166166166166166166166266166 3 goldengate 23043 TNIK NM_015028.1 intron -38329 rs7701284 5 145908423 2 two-hit or HapMap 134391 GPR151 NM_194251.2 adjacent to_5UTR -32554 rs7737236 5 116663689 2 two-hit or HapMap 728342 LOC728342 677_157776157776157776157776157776157776157776157774156777157777159777157_476157777159777159777157476159777157277159776157_776157_776156_776156_776157_776157_776157_776157_776157_776_2_777_2_776157_776157_777 goldengate 57492 ARID1B NM_020732.2 adjacent to_5UTR -618806 rs7801303 7 40884595 2 two-hit or HapMap 79783 C7orfl0 NM_024728.1 adjacent to_3UTR -17713 rs7842021 8 23135149 3 goldengate 8797-NMR303 8786 -186163 np3166-np3306-np3306-np3306-np3306-np3306-np3306-np3306-np3306-np3306-np3386-np3386-np3386-np3309-np3306166 729183 LOC729183 XM_001129594.1 adjacent to_5UTR -14559  SNP Cr Coord 1 BV Gen Name Gen Access Number Location LRG rs7930843 1] 109456617 2 two-hit or Hap ap 728579 LOC728579 XM_001127833.1 adjacent to_3UTR -11784 rs7955850 12 6045840 2 two-hit or HapMap 7450 VWF NM_000552.3 intron -11 rs7970928 12 69902492 2 two-hit or HapMap TSPAN8 NM_004616.2 7103 -64446 adjacent a_5UTR rs798332 7 77746864 3 9863 MAGI2 goldengate NM_012301.3 rs7989372 13 40819933 -23030 intron 3 goldengate NARG1L NM_018527.2 79612 intron 13 96739498 rs7999382 -7347 10150 3 goldengate MBNL2 NM_144778.2 intron -12834 rs8006322 14 78724765 2 two-hit or HapMap 9369 NRXN3 NM_138970.2 intron -90670 rs8015746 14 51028062 2 two-hit or HapMap 729335 LOC729335 XM_001133397.19-9499159159159159159159159159159159159159159 -159155 HapMap 728792 LOC728792 XM_001128492.1 intron -1776 rs8051512 16 83338602 2 two-hit or HapMap 9100 USP10 NM_005153.1 intron -1822 rs8083791 18 63596654 3 goldengate 92126 DSEL NMJD32160.2 adjacent 12267 6807126127126127126127127127127127127127127127127127127127127127127127127127127127127127127127127127127127127127127127127127127127121 dengate 2534 FYN NM_153047.1 intron -304 rs8109493 19 14464932 2 two-hit or HapMap 10755 GIPC1 NM_202467.1 intron -208 rs837511 12 123613242 2 two-hit or HapMap 9612 NCOR2 NM_006312.2 adjacent to_519096 LOC730190 XM_001132852.1 adjacent to_3UTR -61180 rs847126 2 176645080 3 goldengate 344191 EVX2 XM_292968.6 adjacent to_3UTR -8001 rs869026 5 138604699 2 two-hit or HapMap 9782 MATR3 NM_199189137137139137136137136133137137133137137136133137139133137139133137139137136133137139133137139133137139133137139133137139133137136133137139137139133137133137139133137137133137137137137137137137137137137137137137117 adjacent to_3UTR -686191 rs879772 3 193928757 3 goldengate 2257 FGF12 NM_004113.3 adjacent to_5UTR -691 rs882169 11 61448706 3 goldengate 728718 LOC728718 XM_001128272.1 adjacent to_5UTR -3659 33039099909099903 8903 8903 89039093909993 14505725 3 goldengate 6533 SLC6A6 NM_003043.2 adjacent to_3UTR -1751 rs910545 22 26276986 3 goldengate 4330 MN1 NM_002430.2 adjacent to_3UTR -197279 rs911064 20 61370750 3 goldengate 55738 ARFGAP 1 NMJ.75609.1 adjacent to_5UTR -3860 rs9322114 6 148333734 2 two-hit or HapMap 23328 SASH1 NM_015278.3 adjacent to_5UTR -371688 rs936056 17 71099379 2 two-hit or HapMap 728272 -99 XM_00112704.1 889389889 2 887389372 3 goldengate 22934 RPIA NM_144563.2 intron -187 rs9390613 6 149166809 3 goldengate 10090 UST NM_005715.1 intron -56303 rs952656 8 124196776 3 goldengate 93594 WDR67 NM_145647.1 intron -4700 rs9568036 13 47869937 3 goldenU 3168357 3578_57 3578_573573 a578 3573_573573 a578 3573_573336 a3 rs9651511 10 125546364 2 two-hit or HapMap 119587 CPXM2 NMJ.98148.1 intron -1118 rs9788864 16 22921056 2 two-hit or HapMap 57478 USP31 NM_020718.2 adjacent to_3UTR -65812 rs9818995 3 84306729p 2 6436659292 two-stop a_5UTR -782098 rs9822040 3 127410451 3 goldengate 10840 ALDH1L1 NM_012190.2 adjacent to_5UTR -28276 rs9842818 3 147550430 2 two-hit or HapMap 440981 LOC440981 XM_496663.3 adjacent to_3UTR -4140932931 41.2 adjacent to_5UTR -449981 rs9869315 3 177037998 3 goldengate 254827 NAALADL2 NM_207015.1 adjacent to_3UTR -31876 rs9978582 21 46312815 3 goldengate 1292 COL6A2 NM_001849.2 adjacent to_5UTR -29655 rs88572 2app-1 rs2723264 12 38938787 2 two-hit or HapMap 120892 LRRK2 NM_198578.2 ntron -762 rs2022991 5 162168159 3 goldengate 5071 PARK2 NM_013988.1 ntron -41229 rs4709517 161759771 2 two-hit or HapMap 509601 6196 -hit or HapMap 135138 PACRG NM_152410.1 ntron 22547 rs3016563 5 162112846 2 two-hit or HapMap 5071 PARK2 NM_013988.1 ntron 13948 rs3733449 'J 30791345 2 wo-hit or HapMap 5622 5NCA NM_000345U 5383383 873 83 golden 83383 50713ARK2 NM_004S62.1 ntron 230 rsl893098 f L62196867 goldengate 50713ARK2 MM_013988.1 ntron 69937  SNP Cr Coord CV BV Gen Name Gen Access Number Location LRG rs6926 162 190 971 3 6642 5071 goldengate PARK2 NM_013988.1 intron 6 161706684 rs6942109 -64 041 3 goldengate PARK2 NM_013988.1 5071 -5457 rs7298930 12 39005767 intron 2 two-hit or HapMap LRRK2 NM_198578.2 120892 intron 6 161732616 -2392 rsll964284 two two-hit or HapMap 5071 PARK2 NM_004562.1 intron -4717 rsll564173 12 39036738 2 two-hit or HapMap 120892 LRRK2 NM_198578.2 intron -482 rs9347654 6 162840827 2 two-hit or HapMap 5071 PARK2 NM_0045196166906166906166906166 6166-6376166166168 5071 PARK2 NM_013988.1 intron -78498 rsl0878246 12 38918366 2 two-hit or HapMap 120892 LRRK2 NM_198578.2 intron -194 rslll75655 12 38909994 2 two-hit or HapMap 120892 LRRK2 NM_198573 2 -328343 2 -328323 HapMap 120892 LRRK2 NM_198578.2 intron -2319 rsll829088 12 39014046 2 two-hit or HapMap 120892 LRRK2 NM_198578.2 intron -990 rsl0878307 12 38958256 2 two-hit or HapMap 120892 LRRK2.26256255 goldengate 135138 PACRG NM_15241 0.1 adjacent to_5UTR -25473 rsl907632 12 38936769 2 two-hit or HapMap 120892 LRRK2 NM_198578.2 intron -548 rs2000594 6 162161095 3 goldengate 5071 PARK2 NM_013988.1 intron -34165 rsl0857057 4 9318253333gate 8337339333333333333333333333-nd339333333-nd333333333-nd3303333333-nd3303333-nd3303333-nd3d3d9d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d9d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d3d2d2d2 175714565 2 two-hit or HapMap 57179 KIAA1191 NM_020444.2 intron -615 rsl2589063 14 88507193 2 two-hit or HapMap 123016 TTC8 NM_198310.2 adjacent to_3UTR -93105 rsl329579 9 81749779 3 golden9 349283139999913999999999999999999999999139999139139913999139139913999139139139999913913913913999139991391391391391399913913913999139991391391399913999139991399913999139139991399913999139999999992999999296's pass people go to a place 2 two-hit or HapMap 9706 ULK2 NM_014683.2 intron -618 rs2862909 13 23999167 3 goldengate 143 PARP4 NM_006437.2 adjacent to_5UTR -14219 rs3764286 16 83298844 2 two-hit or HapMap 9100 USP10 NM_005153.13rd43-3rd3rd3rd golden -323753rd 286676 ILDR1 NM_175924.2 adjacent to_5UTR -3819 rs7146193 14 88516549 2 two-hit or HapMap 123016 TTC8 NM_198310.2 adjacent to_3UTR -102461 rs7218605 17 74323398 2 two-hit or HapMap 57602 USP36 NM_02 intr90-n02 7539016 1 231556122 2 two-hit or HapMap 84451 KIAA1804 NM_032435.1 intron -54 rs761267 20 41896884 2 two-hit or HapMap 728240 LOC728240 XM_001126959.1 adjacent to_5UTR -79534 rs7653784 3 15227p2155 NM5275 2M5275 NM5155155 NM5275 2152155 NM5155 -10691 rs7924316 11 2130023 2 two-hit or HapMap 51214 IGF2AS NM_016412.1 adjacent to_3UTR -3553 rs9382517 6 55586555 3 goldengate 54511 HMGCLL1 NM_019036.1 adjacent to_5UTR -34611 rs5760103 2 225782IF1 2 42582 M3 -11372 rs4865845 5 53913691 2 two-hit or HapMap 112574 SNAG1 NM_052870.1 adjacent to_3UTR -62265 rs7667015 4 42634138 2 two-hit or HapMap 389207 LOC389207 XM_371694.4 intron -25528 rs7830101A5785153A5AAA 878751 28061 rs4583999 6 156527609 2 two-hit or HapMap 57492 ARID1B NM_020732.2 adjacent to_5UTR -613169 rs741365 12 5554582 2 two-hit or HapMap 57101 TMEM16B NM_020373.1 intron -679 rsll235935 2 858001 NM1001001 NM1001001001 NM1001001001 NM1001001 NM1001001 NM1001001001 NM1001001 NM1001001-1 two-stroke 3626.2 intron -378 rsl740377 6 112149811 2 two-hit or HapMap 2534 FYN NM_153048.1 intron -1853 rsll070178 15 37329814 2 two-hit or HapMap 400360 C15orf54 NM_207445.1 adjacent to_5UTR -397 rs8860H 2 1355 -455p 17p-555 NM_007101.2 coding [127/20] rs4813430 20 21484958 3 goldengate 4821 NKX2-2 NM_002509.2 adjacent to_5UTR -42294 rs3128501 9 89518960 3 goldengate 1612 DAPK1 NMJD04938.2 adjacent to_3UTR -5591 rs7105372 hit 87-7645372app 677p5372pp 2 677p5372pp 2 87p5372p2 NM_213618.1 intron -3401 rs6508999 19 47014387 2 two-hit or HapMap 1084 CEACAM3 NM_001815.1 adjacent to_3UTR -6958 rsl532763 11 121010533 2 two-hit or HapMap 6653 SORL1 NM_003105.3 adjacent-two 183 492 499149492 492 HapMap 8932 MBD2 NM_003927.3 adjacent to_3UTR -203121 rs6047472 20 21478788 2 two-hit or HapMap 4821 NKX2-2 NM_002509.2 adjacent to_5UTR -36124  SNP Cr Coord CV BV Gen Name Gen Access Number Location LRG rs6592941 7 51642120 2 two-hit or HapMap 642663 LOC642663 XM_926118.1 adjacent to_3UTR -224163 rs7027515 9 126213030 3 goldengate 5695 PSMB7 NM_002799.2 intron -1422 5rs420782-722-550 2 7750-2650 2 77682207 hit or HapMap 727723 LOC727723 XM_001125292.1 adjacent to_5UTR -6313 rs4072198 3 62187107 2 two-hit or HapMap 5793 PTP G NM_002841.2 intron -4832 rsll753244 6 165013078 2 two-hit or HapMap 7703297MR 7283297_MR 728356_MR 7283297_MR 728356_MR 728356_MR 728356_MR 728356_MR 728356_MR 728356_MR 7283567_MR 728356_MR 7283567 136740077 2 two-hit or HapMap 9162 DGKI NM_004717.2 intron -3084 rs2399095 3 107605293 2 two-hit or HapMap 728784 LOC728784 XM_001128470.1 adjacent to_5UTR -19073 rs4285366 6 8839136 3 go! 5 163173200 2 two-hit or HapMap 391844 LOC391844 XR_016645.1 adjacent to_5UTR -45558 rsl634079 4 150007154 2 two-hit or HapMap 4306 NR3C2 NM_000901.1 adjacent to_5UTR -424181 rsl0153012 15 92684286 2ap-55 784 MCTP2 NM_018349.2 intron -146 rsl934115 9 23093266 3 goldengate 402360 LOC402360 XR_017263.1 adjacent to_5UTR -578629 rsl5S9854 11 83741948 2 two-hit or HapMap 1740 DLG2 NM_001364.2 intron -101966 2pl2B41966 2B4162BB2199 2B4166BB2196 2B4166 BAP-019792 2B4166 NM_005235.1 adjacent to_3UTR -105135 rs4433214 9 120542512 3 goldengate 442434 LOC442434 XR_016936.1 adjacent to_5UTR -99 rsl0244108 7 55119831 3 goldengate 1956 EGFR NM_201282.1 intron -57642 rs7023309 96576795 155632002 3 goldengate 729853 -99 XM_001134256.1 adjacent to_3UTR -117789 rs2056777 3 25515395 3 goldengate 5915 RARB NM_016152.2 intron -2282 rsll927332 3 70689611 3 goldengate 401072 -99 NM_001013679.1 -588306 0308p-08 55055 ZWILCH NM_017975.2 coding [60/45]
Diseño de un ensayo multiplex (Oligonucleotide Pooling Assay; OPA) con los SNPs seleccionados para la validación en la población española Design of a multiplex trial (Oligonucleotide Pooling Assay; OPA) with the SNPs selected for validation in the Spanish population
5 El panel de marcadores seleccionados durante HFCC (Tabla 2) se envió a la empresa IIlumina Inc para efectuar un análisis bioinformático y determinar las secuencias génicas que se sintetizarán para el ensayo de genotipación múltiple (OPA, oligonucleotide pooling assay) .5 The panel of markers selected during HFCC (Table 2) was sent to the company IIlumina Inc to perform a bioinformatic analysis and determine the gene sequences that will be synthesized for the multiple genotyping assay (OPA, oligonucleotide pooling assay).
0 - Genotipación: Se realizó utilizando la tecnología Veracode (ver más arriba) 0 - Genotyping: It was performed using Veracode technology (see above)
Ejemplo 2. Identificación de la pareja de marcadoresExample 2. Identification of the marker pair
Chr5_rs869026 x chr6_rsl0945364 como factor de susceptibilidad genética para la enfermedad tras la realización de un rastreo completo bi-variante de genoma completo y dos tandas de validación. Para identificar parejas de marcadores genéticos vinculados a la enfermedad en estudio se emplearon, sucesivamente, el HFCC (stage I) y dos validaciones independientes. En cada fase se calculó el efecto con la estimación de la Odds ratio (OR) de los diplotipos. Este término inglés de se ha traducido como razón de posibilidades, razón de momios, razón de odds y es el cociente de dos razones: el numerador es la razón de la probabilidad de que un evento suceda y la probabilidad de que no suceda bajo ciertas condiciones y el denominador es la razón de la probabilidad de que dicho evento suceda y la probabilidad de que no suceda bajo las condiciones complementarias. Es una medida de tamaño de efecto. Finalmente se efectuó un análisis conjunto y meta-análisis de las mejores parejas. Inicialmente HFCC seleccionó combinaciones de los marcadores moleculares descritos en la tabla 2 (que son los mejores candidatos encontrados para explicar el riesgo a padecer la enfermedad) . Las dos validaciones independientes posteriores (stage II y III) permitieron depurar todos los falsos positivos. Concretamente, después de dos rondas de validación, tan sólo se detectó una pareja asociada al fenotipo de manera consistente en todas las poblaciones (tabla 3 y Figura 1) . Chr5_rs869026 x chr6_rsl0945364 as a genetic susceptibility factor for the disease after performing a complete bi-variant complete genome scan and two validation batches. To identify pairs of genetic markers linked to the disease under study, successively, the HFCC (stage I) and two independent validations were used. In each phase the effect was calculated with the estimate of the Odds ratio (OR) of the diplomas. This English term has been translated as ratio of possibilities, ratio of odds, odds ratio and is the ratio of two reasons: the numerator is the reason for the probability of an event happening and the probability that it does not happen under certain conditions and the denominator is the reason for the probability that such an event will happen and the probability that it will not happen under the complementary conditions. It is a measure of effect size. Finally, a joint analysis and meta-analysis of the best couples was carried out. Initially, HFCC selected combinations of the molecular markers described in Table 2 (which are the best candidates found to explain the risk of the disease). The two subsequent independent validations (stage II and III) allowed to purify all false positives. Specifically, after two rounds of validation, only one partner associated to the phenotype was detected consistently in all populations (Table 3 and Figure 1).
Más concretamente se detectó que evaluando el efecto conjunto de una combinación diplotipica de los alelos más frecuentes de marcadores rs869026 y rsl0945364 se encontraba una asociación consistente a la enfermedad en las tres series. De hecho observamos que los individuos portadores en homocigosis de alelo T de rs869026 combinado con la presencia en heterocigosis del alelo C de rsl0945364 (diplotipo TT/CT de los marcadores) tienen un 66-72% más de riesgo de padecer la Enfermedad de Parkinson respecto a los no portadores (Tabla 3) . Tabla 3: Efecto (OR) del diplotipo TT/CT en los marcadores rs869026 y rsl0945364 en la Enfermedad de Parkinson. OR: odds ratio. Ci95-i/s: intervalo de confianza inferior y superior al 95%. More specifically, it was found that evaluating the joint effect of a diploma combination of the most frequent alleles of markers rs869026 and rsl0945364 found a consistent association with the disease in the three series. Indeed, we find that individuals carrying homozygous T allele of rs869026 combined with the presence of heterozygous allele C of rsl0945364 (diplotype TT / CT markers) have 66-72% higher risk of Parkinson's Disease respect to non-carriers (Table 3). Table 3: Effect (OR) of the TT / CT diploma on markers rs869026 and rsl0945364 in Parkinson's disease. OR: odds ratio. Ci95-i / s: confidence interval lower and higher than 95%.
Figure imgf000032_0001
Figure imgf000032_0001
El análisis conjunto de las tres series (n=2697) demostró que el efecto observado es altamente significativo desde un punto de vista estadístico (p=2 , 66E-07 ) . El meta- análisis de las tres series (empleando un modelo de efectos aleatorios) también resultó significativo (p=0,036). Los marcadores que confeccionan dicha pareja (rs869026 y rsl0945364) nunca han sido asociados a la enfermedad de Parkinson y su efecto individual o combinado tampoco ha sido descrito previamente. The joint analysis of the three series (n = 2697) showed that the observed effect is highly significant from a statistical point of view (p = 2, 66E-07). The meta-analysis of the three series (using a random effects model) was also significant (p = 0.036). The markers that make this pair (rs869026 and rsl0945364) have never been associated with Parkinson's disease and its individual or combined effect has not been previously described.
Desde un punto de vista funcional el marcador genómico rsl0945364 se encuentra en un desierto génico (aquella región del genoma donde no hay secuencias codificantes conocidas) del brazo largo del cromosoma 6 (6q27) . Por ello, no puede establecerse ninguna hipótesis acerca de la plausibilidad biológica de esta región cromosómica respecto a la enfermedad de Parkinson puesto que se desconoce qué funciones contiene dicha región. Por contra, el marcador rs869026, localizado en 5q31.2, se encuentra en región 5' del gen SILl. Este gen es responsable del Síndrome de Marinesco-Sj ógren y es un candidato muy contundente para los trastornos neurodegenerativos complejos. No obstante es importante indicar que, hasta la fecha, nunca había sido estudiado o asociado este gen para la enfermedad de Parkinson. From a functional point of view the genomic marker rsl0945364 is found in a gene desert (that region of the genome where there are no known coding sequences) of the long arm of chromosome 6 (6q27). Therefore, no hypothesis can be established about the biological plausibility of this chromosomal region with respect to Parkinson's disease since it is unknown what functions this region contains. In contrast, the rs869026 marker, located at 5q31.2, is in the 5 'region of the SILl gene. This gene is responsible for Marinesco-Sj ógren Syndrome and is a very strong candidate for complex neurodegenerative disorders. However, it is important to indicate that, to date, this gene had never been studied or associated for Parkinson's disease.
Ejemplo 3. Evaluación individual de los marcadores seleccionados por HFCC en tres series independientes Example 3. Individual evaluation of the markers selected by HFCC in three independent series
Sabemos que parte de los resultados de HFCC son debidos a la aparición de marcadores genéticos que tienen un efecto individual per se sin ningún efecto sinérgico con otro marcador. Para valorar esta posibilidad en los marcadores identificados por HFCC, construimos matrices con los genotipos y fenotipos para el software PLINK(PLINK: a tool set for whole-genome association and population-based linkage analyses. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, aller J, Sklar P, de Bakker PI, Daly MJ, Sham PC. Am J Hum Genet . 2007 Sep; 81 (3) : 559-75) . El software permite la evaluación genética de múltiples marcadores y la realización de diferentes test genéticos que sirven como medida de heterogeneidad. Inicialmente aplicamos el comando -assoc de PLINK (que efectúa un test de asociación alélica comparando la frecuencia de los marcadores en casos y controles) a cada serie de forma independiente (stage 1,11 y III) y a una matriz compuesta con todos los genotipos e individuos de las tres series (análisis conjunto). De esta forma se efectuó el análisis sistemático del efecto univariante de los 384 marcadores incluidos en el panel de HFCC midiendo las diferencias de frecuencias alélicas entre casos y controles (tabla 4) . Se observaron 66 marcadores que tenían diferencias estadísticamente significativas de frecuencia alélica entre casos y controles (p<0.05) . No obstante, no es suficiente que observemos asociación en el análisis combinado (tabla 4) sino que el efecto observado en las tres series ha de ser consistente (en la misma dirección la OR) . Para evaluar de manera pormenorizada la ausencia de heterogeneidad entre los tres estudios empleamos diferentes estrategias, i) Los SNPs deben mantener su asociación cuando efectuamos el cálculo de su efecto con una regresión logística ajustada por edad y sexo, ii) deben soportar un análisis estratificado por estudio (Stages 1,2,3) iii) debe existir ausencia de heterogeneidad (índice de Breslow > 0,05) y iv) el meta-análisis ajustado por edad y sexo debe ser positivo. We know that part of the HFCC results are due to the appearance of genetic markers that have an individual effect per se without any synergistic effect with another marker. To assess this possibility in the markers identified by HFCC, build matrices with genotypes and phenotypes for PLINK software (PLINK: a tool set for whole-genome association and population-based linkage analyzes Purcell S, Neale B, Todd-Brown K. , Thomas L, Ferreira MA, Bender D, aller J, Sklar P, from Bakker PI, Daly MJ, Sham PC. Am J Hum Genet. 2007 Sep; 81 (3): 559-75). The software allows the genetic evaluation of multiple markers and the performance of different genetic tests that serve as a measure of heterogeneity. Initially we applied the -assoc PLINK command (which performs an allelic association test comparing the frequency of the markers in cases and controls) to each series independently (stage 1,11 and III) and to a composite matrix with all genotypes and individuals of the three series (joint analysis). In this way, the systematic analysis of the univariate effect of the 384 markers included in the HFCC panel was measured by measuring allelic frequency differences between cases and controls (Table 4). 66 markers were observed that had statistically significant allele frequency differences between cases and controls (p <0.05). However, it is not enough that we observe association in the combined analysis (table 4) but the effect observed in the three series must be consistent (in the same direction the OR). To evaluate in detail the absence of heterogeneity between the three studies we use different strategies, i) SNPs must maintain their association when we calculate their effect with a logistic regression adjusted for age and sex, ii) they must support an analysis stratified by study (Stages 1,2,3) iii) there must be absence of heterogeneity (Breslow index> 0.05) and iv) the meta-analysis adjusted for age and sex must be positive.
Por tanto, para depurar aún más los resultados se procedió al ajuste de efectos individuales de cada SNP en estudio por edad y sexo mediante el comando -logistic, al cálculo de índice de Breslow-Day mediante el comando --within y meta- análisis (—meta-analysis) de los 384 marcadores seleccionados. Gracias a este método combinado identificamos un total de 20 marcadores SNPs que cumplen todos los requisitos de asociación prefijados. Así estos SNPs mostraban significación estadística en el análisis conjunto ajustado por edad y sexo (p<0,05), asociación estadísticamente significativa en el análisis estratificado por series (p<0,05), test de Breslow-Day neutro (no significativo, p>0,05) y meta-análisis ajustado significativo (p<0,05). La aplicación de estos requisitos estadísticos permite reducir la aparición de falsos positivos.  Therefore, in order to further purify the results, the individual effects of each SNP under study by age and sex were adjusted using the -logistic command, the calculation of the Breslow-Day index using the --within and meta-analysis command ( —Meta-analysis) of the 384 selected markers. Thanks to this combined method we identify a total of 20 SNPs markers that meet all the pre-defined association requirements. Thus, these SNPs showed statistical significance in the joint analysis adjusted for age and sex (p <0.05), statistically significant association in the stratified analysis by series (p <0.05), neutral Breslow-Day test (not significant, p> 0.05) and significant adjusted meta-analysis (p <0.05). The application of these statistical requirements reduces the occurrence of false positives.
La tabla 5 muestra los marcadores SNPs seleccionados por HFCC y que se han podido validar analizando dos series independientes adicionales de casos y controles aplicando distintos métodos estadísticos. Destacamos que los marcadores localizados en el cromosoma 12 (rslll75655, rsll564173, rsll564203, rsll829088, rsl907632, rs2723264) se encuentran en el gen LRRK2. Este gen ha sido previamente implicado en PD (9). No obstante su inclusión en la selección efectuada por HFCC demuestra la eficiencia del método aplicado. El resto de los marcadores seleccionados, incluidos en la tabla 4 (rsl039189, rsl2987286, rsl364658, rsl549355, rs218280, rs2332631, rs353111, rs3936139, rs4490160, rs577562, rs6805546, rs761267, rs7650598, rs869026), nunca se habían asociado a PD y son completamente novedosos (tabla 4). De esta manera, se puede calcular la probabilidad de padecer la enfermedad de Parkinson en un individuo problema mediante: Table 5 shows the SNPs markers selected by HFCC and which have been validated by analyzing two additional independent series of cases and controls using different statistical methods. We emphasize that the markers located on chromosome 12 (rslll75655, rsll564173, rsll564203, rsll829088, rsl907632, rs2723264) are found in the LRRK2 gene. This gene has been previously involved in PD (9). However, its inclusion in the selection made by HFCC demonstrates the efficiency of the method applied. The rest of the selected markers, included in table 4 (rsl039189, rsl2987286, rsl364658, rsl549355, rs218280, rs2332631, rs353111, rs3936139, rs4490160, rs577562, rs6805546, rs76598, rs761226, rs761226, rs76126 completely new (table 4). In this way, the probability of suffering from Parkinson's disease in a problem individual can be calculated by:
1. Extracción de una muestra de sangre o cualquier otro tejido para realizar una extracción de ADN a partir de dicho material.  1. Extraction of a blood sample or any other tissue to perform DNA extraction from said material.
2. Una vez obtenido el ADN se procede a genotipar los marcadores identificados mediante tecnologías de genotipado convencionales com PCR-RFLP, ARMS, PCR en tiempo real o secuenciación directa de productos de PCR etc.  2. Once the DNA is obtained, the markers identified by conventional genotyping technologies such as PCR-RFLP, ARMS, real-time PCR or direct sequencing of PCR products etc. are genotyped.
3. Una vez decodificados los genotipos se determina el número de copias del alelo de menor frecuencia que contiene cada individuo y se realiza la suma de estos alelos (cada marcador debe ser ponderados mediante la tabla de coeficientes estandarizados que se suministra)  3. Once the genotypes are decoded, the number of copies of the less frequent allele contained in each individual is determined and the sum of these alleles is performed (each marker must be weighted using the table of standardized coefficients provided)
Dependiendo de la puntuación obtenida podemos conocer si el patrón de genotipos del individuo se parece a lo observado en casos o controles.  Depending on the score obtained we can know if the genotype pattern of the individual resembles what was observed in cases or controls.
Tabla 4 . Análisis sistemático del efecto univariante de los 384 marcadores incluidos en el panel de HFCC midiendo las diferencias de frecuencias alélicas entre casos y controles ID: código de identificación de I Ilumina. CR: cromosoma. CHISQ stagel: valor de Chi-cuadrado obtendi en el estadio 1 de validación. Pstagel: valor de p en estadio 1. ORstagel: efecto (OR) en estadio 1. Idem para estadio 2 y 3 de validación. P conjunta: valor de significación de los tres 2012/000039 Table 4 Systematic analysis of the univariate effect of the 384 markers included in the HFCC panel measuring the allele frequency differences between cases and controls ID: Identification code of I Illuminates. CR: chromosome. CHISQ stagel: Chi-square value obtained in stage 1 of validation. Pstagel: p value in stage 1. ORstagel: effect (OR) in stage 1. Idem for stage 2 and 3 validation. P joint: significance value of the three 2012/000039
34 estudios tomados conjuntamente. ORconjunta: efecto promedio de cada marcador teniendo en cuenta los tres estudios efectuados . 34 studies taken together. ORconjunta: average effect of each marker taking into account the three studies carried out.
Id CR SNP Posición CHISQ P stagel OR CHISQ P stage2 OR CHISQ P OR CHISQ P OR Id CR SNP Position CHISQ P stagel OR CHISQ P stage2 OR CHISQ P OR CHISQ P OR
Stagel stagel stage2 Stage2 Stage3 stage3 stage3 Conjunta conjunta conjunta Stagel stagel stage2 Stage2 Stage3 stage3 stage3 Joint joint joint
225 10 rs2505513 42953543 15,97 6.435E-05 1,81 0,85 0,3565523 0,9 0,22 0,64 1,06 18,7 l,5E-05 1,307225 10 rs2505513 42953543 15.97 6.435E-05 1.81 0.85 0.3565523 0.9 0.22 0.64 1.06 18.7 l, 5E-05 1,307
100 4 Γ52242330 68129844 21,61 3,341E-06 2 0,48 0,4884223 1,09 1,26 0,26 1,13 14,72 0,00012 1,305100 4 Γ52242330 68129844 21.61 3,341E-06 2 0.48 0.4884223 1.09 1.26 0.26 1.13 14.72 0.00012 1.305
44 2 rsl2987286 148380038 9,77 0,0017738 1,51 1,64 0,2003256 1,14 4,97 0,03 1,22 14,3 0,00016 1,25244 2 rsl2987286 148380038 9.77 0.0017738 1.51 1.64 0.2003256 1.14 4.97 0.03 1.22 14.3 0.00016 1.252
43 2 rsl364658 148324278 9,67 0,001873 1,51 1,25 0,2635527 1,12 4,86 0,03 1,22 13,56 0,00023 1,24443 2 rsl364658 148324278 9.67 0.001873 1.51 1.25 0.2635527 1.12 4.86 0.03 1.22 13.56 0.00023 1,244
326 16 rs4888984 78066835 19,64 9.349E-06 2,07 1,74 0,1871389 1,19 0,6 0,44 1,09 13,5 0,00024 1,309326 16 rs4888984 78066835 19.64 9.349E-06 2.07 1.74 0.18871389 1.19 0.6 0.44 1.09 13.5 0.00024 1.309
310 14 rsl2434638 89679833 7,06 0,0078824 0,72 0,16 0,6891565 0,96 13,01 0,00031 0,7676310 14 rsl2434638 89679833 7.06 0.0078824 0.72 0.16 0.68891565 0.96 13.01 0.00031 0.7676
354 19 rs3936139 2538576 10,58 0,0011432 0,65 1,14 0,2856526 0,9 2,8 0,09 0,86 12,62 0,00038 0,8085354 19 rs3936139 2538576 10.58 0.0011432 0.65 1.14 0.28856526 0.9 2.8 0.09 0.86 12.62 0.00038 0.8085
224 10 rsll008264 31129838 6,44 0,0111579 1,41 11,34 0,0007586 1,44 0,1 0,75 1,03 12,37 0,00044 1,244224 10 rsll008264 31129838 6.44 0.0111579 1.41 11.34 0.0007586 1.44 0.1 0.75 1.03 12.37 0.00044 1.244
301 14 rs2332631 25562376 7,45 0,0063436 0,65 9,35 0,0022299 0,69 0,69 0,41 0,92 12,22 0,00047 0,7846301 14 rs2332631 25562376 7.45 0.0063436 0.65 9.35 0.0022299 0.69 0.69 0.41 0.92 12.22 0.00047 0.7846
202 9 rs2381628 7508656 1,99 0,1583409 0,81 0 1 1,01 0,01 0,93 1,01 11,65 0,00064 0,8172202 9 rs2381628 7508656 1.99 0.1583409 0.81 0 1 1.01 0.01 0.93 1.01 11.65 0.00064 0.8172
39 2 rsl370699 86025785 14,07 0,0001761 1,68 2,56 0,1095986 1,18 1,7 0,19 1,13 10,62 0,00112 1,22239 2 rsl370699 86025785 14.07 0.0001761 1.68 2.56 0.1095986 1.18 1.7 0.19 1.13 10.62 0.00112 1,222
96 4 rsl7714109 28900684 8,8 0,0030123 1,61 9,7 0,0018427 1,46 0,09 0,77 1,03 10,48 0,00121 1,26496 4 rsl7714109 28900684 8.8 0.0030123 1.61 9.7 0.0018427 1.46 0.09 0.77 1.03 10.48 0.00121 1,264
239 11 rsl397053 56267270 12,84 0,0003393 0,59 2,88 0,089686 0,83 0,01 0,92 0,99 10,03 0,00154 0,8111239 11 rsl397053 56267270 12.84 0.0003393 0.59 2.88 0.089686 0.83 0.01 0.92 0.99 10.03 0.00154 0.8111
129 5 rs869026 138604699 7,69 0,0055528 0,69 2,65 0,1035499 0,85 0,85 0,36 0,92 10 0,00156 0,8336129 5 rs869026 138604699 7.69 0.0055528 0.69 2.65 0.1035499 0.85 0.85 0.36 0.92 10 0.00156 0.8336
8 1 rs577562 67038563 3,02 0,0822435 0,74 0,7 0,4027837 0,9 2,25 0,13 0,79 9,999 0,00157 0,76488 1 rs577562 67038563 3.02 0.0822435 0.74 0.7 0.4027837 0.9 2.25 0.13 0.79 9.999 0.00157 0.7648
260 12 rslll75655 38909994 4,87 0,0273275 1,47 4,96 0,0259402 1,38 1,2 0,27 1,15 9,913 0,00164 1,297260 12 rslll75655 38909994 4.87 0.0273275 1.47 4.96 0.0259402 1.38 1.2 0.27 1.15 9.913 0.00164 1.297
105 4 rsl828034 148309936 11,34 0,0007586 1,75 0,24 0,6242061 1,08 0,63 0,43 1,1 9,585 0,00196 1,279105 4 rsl828034 148309936 11.34 0.0007586 1.75 0.24 0.6242061 1.08 0.63 0.43 1.1 9.585 0.00196 1.279
99 4 rs3775866 68126775 19,97 7,867E-06 1,87 0,1 0,7518296 1,03 1,78 0,18 1,13 9,548 0,002 1,20899 4 rs3775866 68126775 19.97 7.867E-06 1.87 0.1 0.7518296 1.03 1.78 0.18 1.13 9.548 0.002 1.208
38 2 rsll691934 86018954 16 6,334E-05 1,66 3,13 0,0768638 1,19 0,54 0,46 1,07 9,452 0,00211 1,19138 2 rsll691934 86018954 16 6.334E-05 1.66 3.13 0.0768638 1.19 0.54 0.46 1.07 9,452 0.00211 1,191
90 3 rs7650598 172622161 0,58 0,4463123 1,12 2,02 0,1552392 1,17 7,5 0,01 1,3 8,971 0,00274 1,21190 3 rs7650598 172622161 0.58 0.4463123 1.12 2.02 0.1552392 1.17 7.5 0.01 1.3 8.971 0.00274 1.211
197 8 rs2736000 127469169 14,27 0,0001584 0,58 0,91 0,3401144 0,9 0,45 0,5 0,93 8,947 0,00278 0,821197 8 rs2736000 127469169 14.27 0.0001584 0.58 0.91 0.3401144 0.9 0.45 0.5 0.93 8.947 0.00278 0.821
268 12 rsll564173 39036738 5,45 0,0195683 1,57 1,49 0,2222166 1,19 2,59 0,11 1,21 8,895 0,00286 1,277268 12 rsll564173 39036738 5.45 0.0195683 1.57 1.49 0.2222166 1.19 2.59 0.11 1.21 8.895 0.00286 1.277
45 2 rs353111 165845275 3,53 0,0602678 0,77 8,21 0,004166 0,73 0,55 0,46 0,93 8,877 0,00289 0,832445 2 rs353111 165845275 3.53 0.0602678 0.77 8.21 0.004166 0.73 0.55 0.46 0.93 8.877 0.00289 0.8324
60 3 rs7653784 14848798 10,83 0,0009987 1,6 3,72 0,0537644 1,23 0,36 0,55 1,06 8,55 0,00346 1,20360 3 rs7653784 14848798 10.83 0.0009987 1.6 3.72 0.0537644 1.23 0.36 0.55 1.06 8.55 0.00346 1.203
262 12 rsl907632 38936769 4,56 0,0327271 1,42 1,81 0,1785083 1,19 3,49 0,06 1,24 8,334 0,00389 1,243262 12 rsl907632 38936769 4.56 0.0327271 1.42 1.81 0.1785083 1.19 3.49 0.06 1.24 8.334 0.00389 1,243
263 12 rs2723264 38938787 1,83 0,1761276 0,82 0,03 0,8624902 1,02 6,5 0,01 0,77 8,265 0,00404 0,8258263 12 rs2723264 38938787 1.83 0.1761276 0.82 0.03 0.8624902 1.02 6.5 0.01 0.77 8.265 0.00404 0.8258
261 12 rsl0878246 38918366 0,77 0,3802171 1,17 4,56 0,0327271 1,29 3,03 0,08 1,2 7,79 0,00526 1,221261 12 rsl0878246 38918366 0.77 0.3802171 1.17 4.56 0.0327271 1.29 3.03 0.08 1.2 7.79 0.00526 1.221
34 2 rs4490160 38582415 8,26 0,0040528 1,65 0,97 0,3246802 1,13 2,8 0,09 1,21 7,719 0,00547 1,23334 2 rs4490160 38582415 8.26 0.0040528 1.65 0.97 0.3246802 1.13 2.8 0.09 1.21 7.719 0.00547 1,233
128 5 rs6596293 135544606 15,1 0,000102 1,68 2,6 0,1068637 1,18 0,16 0,69 1,04 7,498 0,00618 1,178128 5 rs6596293 135544606 15.1 0.000102 1.68 2.6 0.1068637 1.18 0.16 0.69 1.04 7,498 0.00618 1,178
269 12 rsll564252 39100000 4,88 0,0271696 1,43 0,57 0,4502589 1,18 3,27 0,07 1,21 7,346 0,00672 1,242269 12 rsll564252 39100000 4.88 0.0271696 1.43 0.57 0.4502589 1.18 3.27 0.07 1.21 7.346 0.00672 1,242
146 6 rsl549355 124849888 2,56 0,1095986 1,27 2,2 0,1380108 1,19 2,18 0,14 1,16 7,307 0,00687 1,203146 6 rsl549355 124849888 2.56 0.1095986 1.27 2.2 0.1380108 1.19 2.18 0.14 1.16 7.307 0.00687 1.203
74 3 rsll715563 83873910 12,44 0,0004202 1,57 1,1 0,2942663 1,11 0,4 0,53 1,07 7,203 0,00728 1,18874 3 rsll715563 83873910 12.44 0.0004202 1.57 1.1 0.2942663 1.11 0.4 0.53 1.07 7.203 0.00728 1,188
351 18 rsl039189 49731452 7,11 0,0076655 0,66 0,21 0,6467674 0,95 2,93 0,09 0,85 6,84 0,00891 0,842351 18 rsl039189 49731452 7.11 0.0076655 0.66 0.21 0.6467674 0.95 2.93 0.09 0.85 6.84 0.00891 0.842
47 2 rs847126 176645080 16,45 4,995E-05 0,54 7,37 0,0066321 0,74 0,55 0,46 1,07 6,79 0,00917 0,847947 2 rs847126 176645080 16.45 4,995E-05 0.54 7.37 0.0066321 0.74 0.55 0.46 1.07 6.79 0.00917 0.8479
143 5 rsl740377 112149811 12,42 0,0004248 1,69 1 0,58 0,45 1,08 6,726 0,0095 1,239143 5 rsl740377 112149811 12.42 0.0004248 1.69 1 0.58 0.45 1.08 6.726 0.0095 1.239
75 i rs4396907 83989494 15 0,000107S 1,7 0,01 0,9203443 3,99 0,39 3,53 1,06 6,693 3,00968 1,17375 i rs4396907 83989494 15 0.000107S 1.7 0.01 0.9203443 3.99 0.39 3.53 1.06 6.693 3.00968 1.173
203 9 rs7023487 16142308 11,23 0,0008049 0,66 1,71 D,1909855 3,88 0,51 0,47 0,94 6,612 3,01013 3,8655
Figure imgf000037_0001
2012/000039 203 9 rs7023487 16142308 11.23 0.0008049 0.66 1.71 D, 1909855 3.88 0.51 0.47 0.94 6.612 3.01013 3.8655
Figure imgf000037_0001
2012/000039
36 36
Figure imgf000038_0001
Figure imgf000039_0001
P T/ES2012/000039
Figure imgf000038_0001
Figure imgf000039_0001
PT / ES2012 / 000039
38 38
Figure imgf000040_0001
Figure imgf000041_0001
2012/000039
Figure imgf000040_0001
Figure imgf000041_0001
2012/000039
40 40
Id CR SNP Posición CHISQ P stagel OR CHISQ P stage2 OR CHISQ P OR CHISQ P OR  Id CR SNP Position CHISQ P stagel OR CHISQ P stage2 OR CHISQ P OR CHISQ P OR
Stagel stagel Stage2 Stage2 Stage3 stage3 stage3 Conjunta conjunta conjunta Stagel stagel Stage2 Stage2 Stage3 stage3 stage3 Joint joint joint
3 1 rsl2119878 9141308 2 0,1572993 0,83 0,12 0,7290345 0,96 0,03 0,87 1,01 0,4794 0,4887 0,95973 1 rsl2119878 9141308 2 0.1572993 0.83 0.12 0.7290345 0.96 0.03 0.87 1.01 0.4794 0.4887 0.9597
366 20 rs6021668 50110836 1,86 0,1726249 1,28 2,46 0,1167788 1,22 0,69 0,41 0,91 0,4787 0,489 1,054366 20 rs6021668 50110836 1.86 0.1726249 1.28 2.46 0.1677788 1.22 0.69 0.41 0.91 0.4778 0.449 1.054
311 14 rs3752958 99252440 3,41 0,0648025 1,4 0,18 0,6713732 0,94 0,33 0,57 1,08 0,4745 0,4909 1,058311 14 rs3752958 99252440 3.41 0.0648025 1.4 0.18 0.6713732 0.94 0.33 0.57 1.08 0.4745 0.4909 1.058
278 12 rsl527101 77063452 1,31 0,2523948 0,87 0,09 0,7641772 1,03 0,65 0,42 0,93 0,4714 0,4923 0,9619278 12 rsl527101 77063452 1.31 0.2223948 0.87 0.09 0.7641772 1.03 0.65 0.42 0.93 0.4714 0.4923 0.9619
342 18 rsl675247 3608506 3,37 0,0663938 1,25 0,31 0,5776802 0,95 2,35 0,13 0,88 0,4708 0,4926 0,9628342 18 rsl675247 3608506 3.37 0.0663938 1.25 0.31 0.5776802 0.95 2.35 0.13 0.88 0.4708 0.4926 0.9628
82 3 rs2953676 115765942 5,36 0,0206038 1,34 1,77 0,1833821 0,87 0 0,99 1 0,4685 0,4937 1,0482 3 rs2953676 115765942 5.36 0.0206038 1.34 1.77 0.1833821 0.87 0 0.99 1 0.4685 0.4937 1.04
276 12 rs7970928 69902492 1,49 0,2222166 1,16 0,01 0,9203443 0,99 0,18 0,67 1,04 0,4616 0,4969 1,038276 12 rs7970928 69902492 1.49 0.2222166 1.16 0.01 0.9203443 0.99 0.18 0.67 1.04 0.4616 0.4969 1,038
153 6 rs4583999 156527609 0,06 0,8064959 0,97 2,69 0,1009798 1,22 6,39 0,01 0,77 0,46 0,4976 0,9554153 6 rs4583999 156527609 0.06 0.8064959 0.97 2.69 0.1009798 1.22 6.39 0.01 0.77 0.46 0.4976 0.9554
316 15 rsl267658 81011209 3,92 0,0477149 0,78 2,06 0,1512102 1,15 0,19 0,66 0,96 0,447 0,5038 0,9629316 15 rsl267658 81011209 3.92 0.0477149 0.78 2.06 0.1512102 1.15 0.19 0.66 0.96 0.477 0.5038 0.9629
323 16 rsl366545 76022537 0,33 0,5656591 0,93 0,12 0,7290345 1,03 0,75 0,39 0,91 0,4461 0,5042 1,042323 16 rsl366545 76022537 0.33 0.5656591 0.93 0.12 0.7290345 1.03 0.75 0.39 0.91 0.4461 0.5042 1.042
152 6 rs7747934 156521972 0,27 0,6033318 0,93 2,26 0,1327541 1,19 5,01 0,03 0,8 0,4375 0,5083 0,9571152 6 rs7747934 156521972 0.27 0.6033318 0.93 2.26 0.1357541 1.19 5.01 0.03 0.8 0.4375 0.5083 0.9571
296 13 rs2057525 92755817 7,44 0,006379 1,49 0,01 0,9203443 1,01 0,29 0,59 0,95 0,435 0,5095 1,045296 13 rs 2057525 92755817 7.44 0.006379 1.49 0.01 0.9203443 1.01 0.29 0.59 0.95 0.435 0.5095 1.045
151 6 rs588036 150740367 0,02 0,8875371 0,98 0,05 0,8230633 0,98 0,86 0,35 1,08 0,4324 0,5108 1,037151 6 rs588036 150740367 0.02 0.8875371 0.98 0.05 0.8230633 0.98 0.86 0.35 1.08 0.4324 0.5108 1.037
1 1 rsl725265 7480780 0,3 0,5838824 0,94 0,3 0,5838824 0,95 1,06 0,3 0,92 0,4281 0,5129 0,96471 1 rsl725265 7480780 0.3 0.5838824 0.94 0.3 0.5838824 0.95 1.06 0.3 0.92 0.4281 0.5129 0.9647
73 3 rs9855978 80171732 2,88 0,089686 1,3 0,01 0,9203443 1,01 0,03 0,86 0,98 0,4276 0,5132 1,04673 3 rs9855978 80171732 2.88 0.089686 1.3 0.01 0.9203443 1.01 0.03 0.86 0.98 0.4276 0.5132 1.046
295 13 rs4142602 92733503 7,4 0,0065224 1,49 0,13 0,718432 1,04 0,68 0,41 0,92 0,3938 0,5303 1,042295 13 rs4142602 92733503 7.4 0.0065224 1.49 0.13 0.718432 1.04 0.68 0.41 0.92 0.3938 0.5303 1,042
343 18 rs617206 6975631 1,63 0,2017031 1,17 0,01 0,9203443 1,01 4,68 0,03 0,83 0,392 0,5312 0,9656343 18 rs 617 206 6975631 1.63 0.2017031 1.17 0.01 0.9203443 1.01 4.68 0.03 0.83 0.392 0.5312 0.9656
383 22 rs733182 41984895 9,83 0,0017169 0,6 0,53 0,4666069 1,09 1,58 0,21 1,15 0,391 0,5318 0,9564383 22 rs733182 41984895 9.83 0.0017169 0.6 0.53 0.4666069 1.09 1.58 0.21 1.15 0.391 0.5318 0.9564
255 12 rsll4305 5118252 1,22 0,2693608 0,86 1,26 0,2616513 1,12 0,9 0,34 1,09 0,3821 0,5365 1,038255 12 rsll4305 5118252 1.22 0.2693608 0.86 1.26 0.2616131 1.12 0.9 0.34 1.09 0.3821 0.5365 1.038
373 21 rs2834684 35234217 0,53 0,4666069 1,1 0,01 0,9203443 0,99 0 0,94 1,01 0,3781 0,5386 1,036373 21 rs2834684 35234217 0.53 0.4666069 1.1 0.01 0.9203443 0.99 0 0.94 1.01 0.3781 0.5386 1.036
277 12 rs2242383 75962738 0,02 0,8875371 0,98 0,87 0,3509553 1,09 0,02 0,88 1,01 0,3711 0,5424 1,035277 12 rs2242383 75962738 0.02 0.8875371 0.98 0.87 0.3509553 1.09 0.02 0.88 1.01 0.3711 0.5424 1.035
320 16 rs9788864 22921056 0,02 0,8875371 0,98 1,32 0,2505922 1,12 0,28 0,6 0,96 0,3711 0,5424 1,035320 16 rs9788864 22921056 0.02 0.8875371 0.98 1.32 0.2505922 1.12 0.28 0.6 0.96 0.3711 0.5424 1.035
291 13 rs2761843 50191038 8,87 0,002899 1,62 0,07 0,7913368 0,97 0,73 0,39 0,91 0,3553 0,5511 1,045291 13 rs2761843 50191038 8.87 0.002899 1.62 0.07 0.7913368 0.97 0.73 0.39 0.91 0.3553 0.5511 1.045
286 13 rs207620 32011403 1,15 0,2835494 1,17 0,16 0,6891565 0,96 1,53 0,22 1,13 0,3535 0,5521 1,038286 13 rs207620 32011403 1.15 0.2835494 1.17 0.16 0.6891565 0.96 1.53 0.22 1.13 0.3535 0.5521 1.038
126 5 rs256996 114943989 1,79 0,1809263 1,18 4,79 0,0286254 1,23 0,03 0,86 0,99 0,3503 0,5539 1,033126 5 rs256996 114943989 1.79 0.1809263 1.18 4.79 0.0286254 1.23 0.03 0.86 0.99 0.3503 0.5539 1.033
67 3 rs4432612 43675296 1,95 0,1625869 0,79 0,42 0,516937 1,09 0,46 0,5 0,93 0,3493 0,5545 0,957967 3 rs4432612 43675296 1.95 0.1625869 0.79 0.42 0.516937 1.09 0.46 0.5 0.93 0.3493 0.5545 0.9579
211 9 rs4407980 35942743 12,71 0,0003637 1,66 0,57 0,4502589 0,92 0,01 0,93 0,99 0,3375 0,5613 1,037211 9 rs4407980 35942743 12.71 0.0003637 1.66 0.57 0.4502589 0.92 0.01 0.93 0.99 0.3375 0.5613 1.037
371 21 rs376691 19278409 3,97 0,0463178 1,29 0,01 0,9203443 0,99 3,79 0,05 0,85 0,3332 0,5638 0,9681371 21 rs376691 19278409 3.97 0.0463178 1.29 0.01 0.9203443 0.99 3.79 0.05 0.85 0.3332 0.5638 0.9681
317 15 rsl0153O12 92684286 1,61 0,2044919 0,83 4,19 0,0406631 1,26 0,39 0,53 1,07 0,3267 0,5676 1,04317 15 rsl0153O12 92684286 1.61 0.2044919 0.83 4.19 0.0406631 1.26 0.39 0.53 1.07 0.3267 0.5676 1.04
66 3 rs4438696 31900672 4,9 0,0268567 0,75 0,69 0,4061644 0,92 1,99 0,16 1,13 0,3142 0,5751 0,967966 3 rs4438696 31900672 4.9 0.0268567 0.75 0.69 0.4061644 0.92 1.99 0.16 1.13 0.3142 0.5751 0.9679
365 20 rs60216S8 50105671 1,5 0,2206715 1,26 2,56 0,1095986 1,23 0,84 0,36 0,9 0,3132 0,5757 1,045365 20 rs60216S8 50 105 671 1.5 0.2206715 1.26 2.56 0.1095986 1.23 0.84 0.36 0.9 0.3132 0.5757 1.045
144 6 rs6907551 114199657 0,85 0,3565523 0,89 0,05 0,8230633 1,02 0,77 0,38 1,08 0,3109 0,5771 1,031144 6 rs6907551 114199657 0.85 0.3565523 0.89 0.05 0.8230633 1.02 0.77 0.38 1.08 0.3109 0.5771 1.031
222 10 rs789 163 18454226 3,38 0,0659921 1,32 0,86 0,3537387 0,9 1,36 0,24 0,89 0,3085 0,5786 0,963222 10 rs789 163 18454226 3.38 0.0659921 1.32 0.86 0.3537387 0.9 1.36 0.24 0.89 0.3085 0.5786 0.963
27 1 rs7539016 231556122 0,57 0,4502589 0,91 1,5 0,2206715 1,13 0,06 0,81 1,02 0,3033 0,5818 1,03127 1 rs7539016 231556122 0.57 0.4502589 0.91 1.5 0.2206715 1.13 0.06 0.81 1.02 0.3033 0.5818 1.031
215 9 Γ51060545 116861439 0,73 0,3928832 0,9 1,29 0,2560481 1,11 2,01 0,16 0,89 0,2928 0,5885 0,9708215 9 Γ51060545 116861439 0.73 0.3928832 0.9 1.29 0.2560481 1.11 2.01 0.16 0.89 0.2928 0.5885 0.9708
171 5 rsl0945364 169193728 0,64 0,4237108 1,11 0,4 0,5270893 1,07 0,51 0,48 0,94 0,2863 0,5926 1,031171 5 rsl0945364 169193728 0.64 0.4237108 1.11 0.4 0.5270893 1.07 0.51 0.48 0.94 0.2863 0.5926 1.031
208 9 rs4495512 29908652 1,25 0,2635527 0,87 0,5 0,4795001 1,07 0,67 0,41 0,93 0,2757 0,5995 0,9699208 9 rs4495512 29908652 1.25 0.2635527 0.87 0.5 0.4795001 1.07 0.67 0.41 0.93 0.2757 0.5995 0.9699
71 3 rslOSHOOl 70715884 3,8 3,0512526 1,35 1,51 0,2191392 0,87 0,11 0,74 0,97 0,2755 0,5997 0,965671 3 rslOSHOOl 70715884 3.8 3.0512526 1.35 1.51 0.2191392 0.87 0.11 0.74 0.97 0.2755 0.5997 0.9656
147 5 rs2749928 131900472 0,13 3,718432 1,05 2,41 0,1205624 0,86 0,08 0,78 1,02 0,2684 0,6044 0,9707147 5 rs2749928 131900472 0.13 3.718432 1.05 2.41 0.1205624 0.86 0.08 0.78 1.02 0.2684 0.6044 0.9707
51 2 rsl035316 224848397 8,98 3,0027295 3,66 3,04 0,8414806 1,02 3,82 3,37 1,09 D,267 0,6054 D,966751 2 rsl035316 224848397 8.98 3.0027295 3.66 3.04 0.8414806 1.02 3.82 3.37 1.09 D, 267 0.6054 D, 9667
243 U S670310 59889222 3,03 3,8624902 1,02 3,83 3,3622725 1,09 3,37 3,54 1,05 0,2665 0,6057 1,03 8 l S3731714 201769065 5,14 ( 3,0132157 3,72 3,03 3,8624902 1,02 3,14 3,71 1,03 3,2653 3,6065 3,9695
Figure imgf000043_0001
Id CR SNP Posición CHISQ P stagel OR CHISQ P stage2 OR CHISQ P OR CHISQ P OR 243 U S670310 59889222 3.03 3.8624902 1.02 3.83 3.3622725 1.09 3.37 3.54 1.05 0.2665 0.6057 1.03 8 l S3731714 2017 69065 5.14 (3, 0132157 3.72 3.03 3.8624902 1.02 3.14 3.71 1.03 3,2653 3,6065 3,9695
Figure imgf000043_0001
Id CR SNP Position CHISQ P stagel OR CHISQ P stage2 OR CHISQ P OR CHISQ P OR
Stagel stagel stage2 Stage2 Stage3 stage3 stage3 Conjunta conjunta conjunta Stagel stagel stage2 Stage2 Stage3 stage3 stage3 Joint joint joint
229 10 rslll96543 115684622 3,05 0,0807372 0,78 0,53 0,4666069 1,09 0 0,99 1 0,06629 0,7968 0,9835229 10 rslll96543 115684622 3.05 0.0807372 0.78 0.53 0.4666069 1.09 0 0.99 1 0.06629 0.7968 0.9835
189 8 rs7842021 23135149 11,2 0,000818 0,66 0,75 0,3864762 0,92 1,81 0,18 0,9 0,06577 0,7976 1,014189 8 rs7842021 23135149 11.2 0.000818 0.66 0.75 0.3864762 0.92 1.81 0.18 0.9 0.06577 0.7976 1,014
367 20 rs6021688 50126404 4,58 0,0323472 1,44 1 0,3173108 1,13 1,78 0,18 0,86 0,06478 0,7991 1,019367 20 rs6021688 50126404 4.58 0.0323472 1.44 1 0.3173108 1.13 1.78 0.18 0.86 0.06478 0.7991 1.019
218 9 126213030 0,62 0,4310473 1,1 0,02 0,8875371 1,01 0,73 0.39 0,93 0,06425 0,7999 1,014218 9 126213030 0.62 0.4310473 1.1 0.02 0.8875371 1.01 0.73 0.39 0.93 0.06425 0.7999 1,014
155 6 rs6942109 161706684 0,63 0,4273553 0,91 2,63 0,1048617 0,85 2,61 0,11 1,15 0,06337 0,8012 0,986155 6 rs6942109 161706684 0.63 0.4273553 0.91 2.63 0.1048617 0.85 2.61 0.11 1.15 0.06337 0.8012 0.986
360 19 rs7254214 47060578 0,01 0,9203443 1,02 0 1 1,01 0,23 0,63 0,96 0,05882 0,8084 0,9868360 19 rs7254214 47060578 0.01 0.9203443 1.02 0 1 1.01 0.23 0.63 0.96 0.05882 0.8084 0.9868
10 1 rsl2144940 99705926 1,67 0,1962586 0,83 0,59 0,4424191 1,08 0,53 0,47 1,07 0,0584 0,809 1,01510 1 rsl2144940 99705926 1.67 0.19962586 0.83 0.59 0.4424191 1.08 0.53 0.47 1.07 0.0584 0.809 1.015
59 3 rs9036 14505725 3,83 0,0503429 0,74 0,89 0,3454773 1,12 0,77 0,38 1,09 0,05752 0,8105 1,01659 3 rs9036 14505725 3.83 0.0503429 0.74 0.89 0.3454773 1.12 0.77 0.38 1.09 0.05752 0.8105 1.016
297 13 rs7999382 96739498 4,11 0,0426304 0,77 1,08 0,2986978 1,11 1,25 0,26 1,1 0,05751 0,8105 1,014297 13 rs7999382 96739498 4.11 0.0426304 0.77 1.08 0.2986978 1.11 1.25 0.26 1.1 0.05751 0.8105 1.014
158 6 rs6906519 161811378 1,84 0,174951 1,25 0,01 0,9203443 1,02 1,31 0,25 0,88 0,05635 0,8124 1,018158 6 rs6906519 161811378 1.84 0.174951 1.25 0.01 0.9203443 1.02 1.31 0.25 0.88 0.05635 0.8124 1.018
114 5 rs324454 55238308 3,6 0,0577796 1,26 1,51 0,2191392 0,89 0,09 0,76 1,03 0,05612 0,8127 0,9871114 5 rs324454 55238308 3.6 0.0577796 1.26 1.51 0.2191392 0.89 0.09 0.76 1.03 0.05612 0.8127 0.9871
240 11 rsl07S0861 56937983 1,62 0,2030919 0,85 4,68 0,0305158 1,24 0,83 0,36 0,93 0,05574 0,8134 1,014240 11 rsl07S0861 56937983 1.62 0.2030919 0.85 4.68 0.0305158 1.24 0.83 0.36 0.93 0.05574 0.8134 1.014
284 13 rs2862909 23999167 0,45 0,502335 0,91 0,15 0,6985354 1,04 0,54 0,46 1,05 0,04872 0,8253 1,012284 13 rs2862909 23999167 0.45 0.502335 0.91 0.15 0.6885354 1.04 0.54 0.46 1.05 0.04872 0.8253 1.012
194 8 rsl0094702 89403625 7,27 0,0070116 0,67 3,41 0,0648025 1,26 0,62 0,43 0,93 0,04256 0,8366 0,986194 8 rsl0094702 89403625 7.27 0.0070116 0.67 3.41 0.0648025 1.26 0.62 0.43 0.93 0.04256 0.8366 0.986
275 12 Γ52470378 69512747 15,02 0,0001064 0,56 5,38 0,0203689 1,28 2,35 0,13 1,17 0,03948 0,8425 1,013275 12 2452470378 69512747 15.02 0.0001064 0.56 5.38 0.0203689 1.28 2.35 0.13 1.17 0.03948 0.8425 1.013
227 10 rs6583661 82553590 1,63 0,2017031 0,84 1,91 0,1669633 0,87 3,04 0,08 1,17 0,0389 0,8436 0,9882227 10 rs6583661 82553590 1.63 0.2017031 0.84 1.91 0.1669633 0.87 3.04 0.08 1.17 0.0389 0.8436 0.9882
183 7 rs2253729 139589358 3,24 0,0718606 1,27 2,88 0,089686 0,84 1,19 0,28 1,1 0,03784 0,8458 1,011183 7 rs2253729 139589358 3.24 0.0718606 1.27 2.88 0.089686 0.84 1.19 0.28 1.1 0.03784 0.8458 1.011
290 13 rs9568036 47869937 3,47 0,0624913 0,79 2,14 0,1435019 0,85 6,68 0,01 1,24 0,03471 0,8522 1,011290 13 rs9568036 47869937 3.47 0.0624913 0.79 2.14 0.1435019 0.85 6.68 0.01 1.24 0.03471 0.8522 1.011
321 16 52912715 0,37 0,5430043 1,08 0,01 0,9203443 1,01 0,06 0,8 1,02 0,03458 0,8525 1,01321 16 52912715 0.37 0.5430043 1.08 0.01 0.9203443 1.01 0.06 0.8 1.02 0.03458 0.8525 1.01
14 1 156562253 10,38 0,0012739 0,67 0 1 1 3,49 0,06 1,17 0,03376 0,8542 0,989614 1 156562253 10.38 0.0012739 0.67 0 1 1 3.49 0.06 1.17 0.03376 0.8542 0.9896
192 8 rs7465573 61311677 2,03 0,1542207 0,77 0,38 0,5376032 1,1 0,12 0,73 1,04 0,02926 0,8642 1,014192 8 rs7465573 61311677 2.03 0.1542207 0.77 0.38 0.5376032 1.1 0.12 0.73 1.04 0.02926 0.8642 1.014
370 20 rs911064 61370750 1,79 0,1809263 1,22 0,02 0,8875371 0,99 0,35 0,55 0,95 0,02896 0,8649 1,011370 20 rs911064 61370750 1.79 0.1809263 1.22 0.02 0.8875371 0.99 0.35 0.55 0.95 0.02896 0.8649 1,011
348 18 rsl365461 34356804 0,06 0,8064959 0,97 0,04 0,8414806 1,02 0,64 0,43 0,94 0,02801 0,8671 0,9908348 18 rsl365461 34356804 0.06 0.8064959 0.97 0.04 0.8414806 1.02 0.64 0.43 0.94 0.02801 0.8671 0.9908
21 1 rs2648774 197382785 1,96 0,1615134 0,84 3,08 0,0792605 1,18 0,08 0,78 0,98 0,02694 0,8696 1,00921 1 rs2648774 197382785 1.96 0.1615134 0.84 3.08 0.0792605 1.18 0.08 0.78 0.98 0.02694 0.8696 1.009
110 4 Γ54861960 182609327 1,28 0,2578992 1,24 0,05 0,8230633 0,97 0,09 0,76 0,96 0,02516 0,874 1,013110 4 4854861960 182609327 1.28 0.2578992 1.24 0.05 0.8230633 0.97 0.09 0.76 0.96 0.02516 0.874 1.013
139 6 rs3094694 30559883 1,07 0,3009457 0,84 0,15 0,6985354 0,95 0,23 0,63 1,05 0,02302 0,8794 0,9887139 6 rs3094694 30559883 1.07 0.3009457 0.84 0.15 0.685354 0.95 0.23 0.63 1.05 0.02302 0.8794 0.9887
11 1 rs724479 102047645 3,54 0,0599055 1,38 0,08 0,7772974 0,97 0,01 0,92 0,99 0,02279 0,88 1,01111 1 rs724479 102047645 3.54 0.0599055 1.38 0.08 0.7772974 0.97 0.01 0.92 0.99 0.02279 0.88 1.011
233 11 rs7924316 2130023 17,44 2,965E-05 1,68 2,2 0,1380108 1,15 1,08 0,3 0,92 0,02193 0,8823 0,9919233 11 rs7924316 2130023 17.44 2,965E-05 1.68 2.2 0.1380108 1.15 1.08 0.3 0.92 0.02193 0.8823 0.9919
359 19 rs6508999 47014387 0,12 0,7290345 0,96 0,15 0,6985354 1,04 0 0,96 1 0,01776 0,894 1,008359 19 rs6508999 47014387 0.12 0.7290345 0.96 0.15 0.6885354 1.04 0 0.96 1 0.01776 0.894 1.008
199 9 1705820 3,22 0,0727436 1,38 1,56 0,2116653 0,83 0,3 0,58 0,94 0,01698 0,8963 0,9894199 9 1705820 3.22 0.0727436 1.38 1.56 0.2116653 0.83 0.3 0.58 0.94 0.01698 0.8963 0.9894
145 6 rs3777981 117841948 5,64 0,0175552 1,34 0,58 0,4463123 0,93 1,33 0,25 0,91 0,01682 0,8968 0,9929145 6 rs3777981 117841948 5.64 0.0175552 1.34 0.58 0.4463123 0.93 1.33 0.25 0.91 0.01682 0.8968 0.9929
6 1 rs2077066 23650949 0,1 0,7518296 1,04 0 1 1 0,1 0,75 0,97 0,01622 0,8987 0,99266 1 rs2077066 23650949 0.1 0.7518296 1.04 0 1 1 0.1 0.75 0.97 0.01622 0.8987 0.9926
376 21 rs6518223 45538977 0,72 0,3961439 1,11 0,1 0,7518296 0,97 0,04 0,83 0,98 0,01557 0,9007 1,007376 21 rs6518223 45538977 0.72 0.3961439 1.11 0.1 0.7518296 0.97 0.04 0.83 0.98 0.01557 0.9007 1.007
274 12 rsl022242 69492622 12,99 0,0003132 0,58 3,91 0,0479996 1,24 1,4 0,24 1,13 0,01522 0,9018 0,9919274 12 rsl022242 69492622 12.99 0.0003132 0.58 3.91 0.0479996 1.24 1.4 0.24 1.13 0.01522 0.9018 0.9919
212 9 rsl329579 81749779 3,95 0,0468714 0,77 0,96 0,3271869 1,1 1,08 0,3 1,1 0,01379 0,9065 1,007212 9 rsl329579 81749779 3.95 0.0468714 0.77 0.96 0.3281869 1.1 1.08 0.3 1.1 0.01379 0.9065 1.007
41 2 rs938372 88780884 2,7 0,1003483 1,25 0,21 0,6467674 0,95 0,84 0,36 0,92 0,01358 0,9072 1,00741 2 rs938372 88780884 2.7 0.1003483 1.25 0.21 0.6467674 0.95 0.84 0.36 0.92 0.01358 0.9072 1.007
81 3 rs2399095 107605293 1,09 0,2964715 0,88 0,72 0,3961439 0,92 0,24 0,62 0,96 0,01357 0,9073 1,00681 3 rs2399095 107605293 1.09 0.2964715 0.88 0.72 0.3961439 0.92 0.24 0.62 0.96 0.01357 0.9073 1.006
116 5 rs286002 55742226 8,12 0,004378 1,51 0 1 1,01 1,64 0,2 0,89 0,01229 0,9117 1,007116 5 rs286002 55742226 8.12 0.004378 1.51 0 1 1.01 1.64 0.2 0.89 0.01229 0.9117 1.007
101 4 rs2869872 77410136 7,98 0,0047297 1,51 5,45 0,0195683 0,77 0,29 0,59 1,06 0,01153 0,9145 1,007101 4 rs2869872 77410136 7.98 0.0047297 1.51 5.45 0.0195683 0.77 0.29 0.59 1.06 0.01153 0.9145 1.007
56 3 rs7649851 5255410 1,05 3,3055074 3,84 0,05 0,8230633 1,03 0,37 0,54 1,07 0,01105 0,9163 1,00856 3 rs7649851 5255410 1.05 3.3055074 3.84 0.05 0.8230633 1.03 0.37 0.54 1.07 0.01105 0.9163 1.008
180 7 rsl2672177 101813787 1,83 3,1761276 3,81 2,93 3,0869475 1,22 0,93 3,34 3,91 D.007073 0,933 3,9943180 7 rsl2672177 101813787 1.83 3.1761276 3.81 2.93 3.0869475 1.22 0.93 3.34 3.91 D.007073 0.933 3.9943
358 19 -s7251154 ' J6997781 3,02 3,8875371 3,98 3,06 3,8064959 1,03 3,03 3,86 3,99 3,004482 3,9466 1,004
Figure imgf000045_0001
358 19 -s7251154 'J6997781 3.02 3.8875371 3.98 3.06 3.8064959 1.03 3.03 3.86 3.99 3.004482 3.9466 1.004
Figure imgf000045_0001
Tabla 5. Marcadores SNPs seleccionados por HFCC y validados analizando dos series independientes adicionales de casos y controles . Table 5. SNPs markers selected by HFCC and validated by analyzing two additional independent series of cases and controls.
Figure imgf000045_0002
Ejemplo 4. Construcción de un Panel Predictivo con los marcadores seleccionados con HFCC
Figure imgf000045_0002
Example 4. Construction of a Predictive Panel with the selected markers with HFCC
Una manera diferente de demostrar la capacidad diagnóstica de los marcadores que selecciona HFCC, es la elaboración de un panel predictivo y la realización con dicho panel de un análisis discriminante, junto el cálculo de sensibilidad y especificidad de dicho panel, mediante la determinación del área bajo la curva que el predictor determina. Aunque resultaría tentador realizar el análisis discriminante usando los 384 SNPs seleccionados por HFCC simultáneamente, ésto no se llevó a cabo. Los motivos son la alta probabilidad de sobreajuste que tienen los análisis discriminantes si introducimos tantas variables, el ruido estadístico que sabemos acarrea el uso de la tecnología multivariante (como demuestra el ejemplo 1) y la existencia de heterogeneidad no alélica que es una característica muy común en las enfermedades complejas. Por todo ello, se decidió adoptar una estrategia más conservadora y seleccionar, para el análisis discriminante, sólo aquellos marcadores que mostraran asociación de algún modo a la patología (PD) . Así, para la construcción del panel empleamos una combinación de SNPs que habíamos asociado al "status" de casos (estos son los marcadores presentes en la tabla 5) a los que añadimos, además, un pequeño subconjunto de marcadores que presentaban indicios estadísticamente significativos de asociación con la edad de comienzo de la enfermedad (AAO, age-at-onset ) (tabla 6) . Los marcadores asociados a AAO son también interesantes pues si bien no están directamente asociados a PD, pueden modificar el curso de la patología acelerando o decelerando el proceso neurodegenerativo y, en consecuencia, afectar al pronóstico de la enfermedad. La tabla 6 es fruto de esta segunda selección de marcadores y aporta un total de 21 SNPs del panel de HFCC que se asocian significativamente (p<0,05) a la edad de aparición de PD (AAO) . Este cálculo se realizó con plink (PLINK: a tool set for whole-genome association and population-based linkage analyses. Purcell S, Neale B, Todd- Brown K, Thomas L, Ferreira MA, Bender D, aller J, Sklar P, de Bakker PI, Daly MJ, Sham PC.Am J Hum Genet . 2007 Sep; 81 (3) : 559-75) empleando la regresión lineal (--linear) ajustando el efecto por sexo y por la serie a la que pertenece el paciente. A different way of demonstrating the diagnostic capacity of the markers that HFCC selects is the development of a predictive panel and the realization with said panel of a discriminant analysis, together with the calculation of sensitivity and specificity of said panel, by determining the low area the curve that the predictor determines. Although it would be tempting to perform the discriminant analysis using the 384 SNPs selected by HFCC simultaneously, this was not carried out. The reasons are the high probability of over-adjustment that discriminant analyzes have if we introduce so many variables, the statistical noise that we know entails the use of multivariate technology (as shown in example 1) and the existence of non-allelic heterogeneity that is a very common characteristic. in complex diseases. Therefore, it was decided to adopt a more conservative strategy and select, for the discriminant analysis, only those markers that showed somehow association with the pathology (PD). Thus, for the construction of the panel we used a combination of SNPs that we had associated with the "status" of cases (these are the markers present in table 5) to which we also added a small subset of markers that presented statistically significant indications of association with the age of onset of the disease (AAO, age-at-onset) (table 6). The markers associated with AAO are also interesting because although they are not directly associated with PD, they can modify the course of the pathology by accelerating or decelerating the neurodegenerative process and, consequently, affecting the prognosis of the disease. Table 6 is the result of this second selection of markers and provides a total of 21 SNPs of the HFCC panel that are significantly associated (p <0.05) to the age of PD onset (AAO). This calculation was carried out with plink (PLINK: a tool set for whole-genome association and population-based linkage analyses. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, aller J, Sklar P, from Bakker PI, Daly MJ, Sham PC.Am J Hum Genet. 2007 Sep; 81 (3): 559-75) using linear regression (--linear) adjusting the effect by sex and by the series to which the patient.
Tabla 6. Combinación de SNPs asociados a la enfermedad con SNPs asociados a la edad de aparición de ésta. Table 6. Combination of SNPs associated with the disease with SNPs associated with their age of onset.
SNP: polimorfismo de un único nucleótido. CHR: cromosoma. BP: pares de bases. Al: alelo 1. NMISS: número de individuos disponibles para el studio. BETA: coeficiente Beta de regresión lineal. Stat: valor estadístico de la regresión lineal. P: valor de significación. SNP: single nucleotide polymorphism. CHR: chromosome. BP: base pairs. Al: allele 1. NMISS: number of individuals available for the study. BETA: Beta linear regression coefficient. Stat: statistical value of the linear regression. P: significance value.
SNP CHR BP Al TEST NMISS BETA STAT P Gen SNP CHR BP Al TEST NMISS BETA STAT P Gen
rs2986574 1 182173237 A ADD 1305 -1,234 -1,964 0,0497 GLT25D2 rsllll9097 1 206813021 G ADD 1303 1,335 2,281 0,0227 desert rs2986574 1 182173237 A ADD 1305 -1.234 -1.964 0.0497 GLT25D2 rsllll9797 1 206813021 G ADD 1303 1.335 2.281 0.0227 desert
rs4658673 1 243108935 A ADD 1301 1,348 2,232 0,0258 HN NPU rsl0496075 2 57762864 C ADD 1306 1,463 2,718 0,0066 BG924755 rs847126 2 176645080 C ADD 1286 -1,262 -2,084 0,0374 EVX2 rs4658673 1 243108935 A ADD 1301 1,348 2,232 0.0258 HN NPU rsl0496075 2 57762864 C ADD 1306 1,463 2,718 0.0066 BG924755 rs847126 2 176645080 C ADD 1286 -1,262 -2,084 0,0374 EVX2
rs9036 3 14505725 C ADD 1309 -1,642 -2,742 0,0062 SLC6A6 rs4505714 3 84573028 G ADD 1285 1,105 1,984 0,0475 desert rs9036 3 14505725 C ADD 1309 -1,642 -2,742 0.0062 SLC6A6 rs4505714 3 84573028 G ADD 1285 1,105 1,984 0.0475 desert
rsl0155062 4 7184806 A ADD 1307 1,168 2,169 0,0302 SORCS2 rs3094694 6 30559883 G ADD 1308 -1,424 -2,104 0,0355 BI226110 rs7747934 6 156521972 C ADD 1307 -1,224 -2,096 0,0363 CB984582 rs4583999 6 156527609 G ADD 1286 -1,166 -1,965 0,0496 CB984582 rsl0945364 6 169193728 T ADD 1309 1,177 2,255 0,0243 desert rsl0155062 4 7184806 A ADD 1307 1,168 2,169 0.0302 SORCS2 rs3094694 6 30559883 G ADD 1308 -1,424 -2,104 0.0355 BI226110 rs7747934 6 156521972 C ADD 1307 -1,224 -2,096 0,0363 CB984582 rs4583609 6 AD65276,999 6 1565,999 -1 1565,965 -166 -165276999 G 15,627,996 -1,665,965 -1,696 665 -199 -1 G 0.0496 CB984582 rsl0945364 6 169193728 T ADD 1309 1,177 2,255 0,0243 desert
rsll56143 7 136740077 T ADD 1308 1,337 2,495 0,0127 DGKI rsll56143 7 136740077 T ADD 1308 1,337 2,495 0,0127 DGKI
rsl888349 7 138314672 T ADD 1309 1,535 2,394 0,0168 KIAA1549 rsl2353255 9 20911332 A ADD 1305 -1,158 -2,124 0,0338 KIAA1797 rs7027515 9 126213030 C ADD 1308 1,2 2,369 0,018 PSMB7 rsl888349 7 138314672 T ADD 1309 1,535 2,394 0.0168 KIAA1549 rsl2353255 9 20911332 A ADD 1305 -1,158 -2,124 0.0338 KIAA1797 rs7027515 9 126213030 C ADD 1308 1.2 2,369 0.018 PSMB7
rs332185 10 28953315 C ADD 1309 :1,207 -2,058 0,0397 BG563864 rs763373 11 12796918 C ADD 1254 1,316 2,465 0,0138 TEAD1 SNP CHR BP Al TEST NMISS BETA STAT P Gen rs332185 10 28953315 C ADD 1309 : 1,207 -2,058 0.0397 BG563864 rs763373 11 12796918 C ADD 1254 1,316 2,465 0.0138 TEAD1 SNP CHR BP Al TEST NMISS BETA STAT P Gen
rsl0750861 11 56937983 C ADD 1308 1,023 2,058 0,0398 SLC43A3 rs8109493 19 14464932 c ADD 1306 -1,245 -1,994 0,0464 GIPC1  rsl0750861 11 56937983 C ADD 1308 1,023 2,058 0.0398 SLC43A3 rs8109493 19 14464932 c ADD 1306 -1,245 -1,994 0,0464 GIPC1
rs6518223 21 45538977 c ADD 1307 1,036 2,082 0,0376 AA307589  rs6518223 21 45538977 c ADD 1307 1,036 2,082 0.0376 AA307589
De manera que para efectuar el análisis discriminante con el software SPSS (Lead technologies Inc) construimos una nueva matriz de marcadores SNPs e incluimos tanto los marcadores contenidos en la tabla 5 como aquellos que aparecen en la tabla 6, la edad, el sexo de los casos y controles y la serie a la que pertenecían (stage 1,2 ó 3). Es importante detallar que se eliminaron aquellos marcadores que resultaban redundantes bien porque aparecían en ambas tablas o bien porque trazaban el mismo gen (como es el caso de las multiplicidades detectadas para LRRK2 o la duplicidad detectada para ACVR2) . La lista de los 34 SNPs empleados en el análisis discriminante se detalla en la tabla 7 junto con los coeficientes que emplea cada uno para la construcción de la función canónica que consiste en la suma ponderada de las variables incluidas en el panel predictivo. Igualmente, incluimos los coeficientes asignados a la edad, el sexo y la serie a la que pertenecen (todos estos coeficientes se calculan con SPSS) . So to perform the discriminant analysis with the SPSS (Lead technologies Inc) software, we build a new matrix of SNPs markers and include both the markers contained in table 5 and those that appear in table 6, the age, the sex of the cases and controls and the series to which they belonged (stage 1,2 or 3). It is important to detail that those markers that were redundant were eliminated either because they appeared in both tables or because they traced the same gene (as is the case with the multiplicities detected for LRRK2 or the duplicity detected for ACVR2). The list of the 34 SNPs used in the discriminant analysis is detailed in Table 7 together with the coefficients that each one uses for the construction of the canonical function consisting of the weighted sum of the variables included in the predictive panel. Likewise, we include the coefficients assigned to the age, sex and series to which they belong (all these coefficients are calculated with SPSS).
Tabla 7 : Coeficientes es andarizados de las funciones discriminantes canónicas Table 7: Coefficients are standardized for canonical discriminant functions
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000048_0001
Figure imgf000049_0001
La tabla 8 muestra los resultados del análisis discriminante y la validación cruzada empleando la función construida y emitiendo una puntuación para cada individuo con los coeficientes de la tabla 7. Los resultados demuestran una capacidad de predicción del estatus de los controles del 72,8% (validación cruzada 71,8%) y una capacidad de predicción del estatus de casos del 77,4% con una validación cruzada resultante del 75,7%. Estas estimaciones exceden el ampliamente el 50% (que es lo que se estima que sucedería si se tratara de predecir la condición de enfermo PD o sano lanzando una moneda al aire (es decir por azar , tabla 8 Table 8 shows the results of the discriminant analysis and cross-validation using the constructed function and issuing a score for each individual with the coefficients of Table 7. The results demonstrate a predictive capacity of the control status of 72.8% ( cross validation 71.8%) and a predictive capacity of case status of 77.4% with a resulting cross validation of 75.7%. These estimates far exceed 50% (which is estimated to happen if it will be about predicting the condition of sick PD or healthy by throwing a coin in the air (that is by chance, table 8
Tabla 8: Resultados de la clasificación (b,c) Table 8: Results of the classification (b, c)
Grupo de pertenencia  Membership Group
case control pronosticado Total  Total forecasted case control
Controles Casos 1  Controls Cases 1
Original Recuento Controles 825 308 1133  Original Count Controls 825 308 1133
Casos 226 773 999  Cases 226 773 999
% Controles 72,8 27,2 100,0  % Controls 72.8 27.2 100.0
Casos 22,6 77,4 100,0  Cases 22.6 77.4 100.0
Validación Recuento Controles  Validation Counting Controls
814 319 1133  814 319 1133
cruzada(a)  crossed
Casos 243 756 999  Cases 243 756 999
% Controles 71,8 28,2 100,0  % Controls 71.8 28.2 100.0
Casos 24,3 75,7 100,0  Cases 24.3 75.7 100.0
a La validación cruzada sólo se aplica a los casos del análisis. En la validación cruzada, cada caso se clasifica mediante las funciones derivadas a partir del resto de los casos. a Cross validation only applies to the cases of the analysis. In cross-validation, each case is classified by the functions derived from the rest of the cases.
b Clasificados correctamente el 75,0% de los casos agrupados originales. b Correctly classified 75.0% of the original grouped cases.
c Clasificados correctamente el 73,6% de los casos agrupados validados mediante validación cruzada. c Correctly classified 73.6% of the grouped cases validated by cross validation.
Para medir el efecto de la adición de los marcadores a las variables edad, sexo y obtener la significación estadística del conjunto de SNPs seleccionados se calculó el área bajo la curva (COR) que determinan per se los SNPs, las co-variables críticas per se (edad, sexo) y ambas en conjunto (Figura 2 y tabla 9) . De nuevo los resultados demuestran que los SNPs seleccionados tienen una capacidad predictiva inherente que excede lo esperable por azar (área 61,4%, p<l,14E-19) y, además mejoran la capacidad predictiva de la co-variables en un 2% aproximadamente (tabla 9) . Tabla 9: Área bajo la curva To measure the effect of adding markers to the variables age, sex and obtaining the statistical significance of the set of selected SNPs, the area under the curve (COR) determined by the SNPs, the critical co-variables per se was calculated. (age, sex) and both together (Figure 2 and table 9). Again, the results show that the selected SNPs have an inherent predictive capacity that exceeds what is expected by chance (area 61.4%, p <l, 14E-19) and also improves the predictive capacity of the co-variables by 2 % approximately (table 9). Table 9: Area under the curve
Figure imgf000051_0001
Figure imgf000051_0001
a Bajo el supuesto no paramétrico a Under the non-parametric assumption
b Hipótesis nula: área verdadera = 0,5 b Null hypothesis: true area = 0.5
Los datos obtenidos con la presente invención indican, por tanto, que la combinación de SNPs rs577562, rs2986574, rsllll9097, rs4658673, rs4490160, rsl0496075, rsl2987286, rs847126, rs9036, rs6805546, rs4505714, rs7650598, rsl0155062, rs218280, rs869026, rs3094694, rsl549355, rs7747934, rs4583999, rsl0945364, rsll56143, rsl888349, rsl2353255, rs7027515, rs332185, rs763373, rsl0750861, rsll564173, rs2332631, rsl039189, rs3936139, rs8109493, rs761267, y rs6518223 analizados con untamente con el algoritmo utilizado en la presente invención puede identificar a un subconjunto de individuos de la población con riesgo de padecer la enfermedad de Parkinson esporádica incluso sin tener en cuenta su género, edad o procedencia. Ejemplo 5. Primera Validación de los marcadores seleccionados (Población Española) The data obtained with the present invention indicate, therefore, that the combination of SNPs rs577562, rs2986574, rsllll9097, rs4658673, rs4490160, rsl0496075, rsl2987286, rs847126, rs9036, rs6805546, rs450575, rs5355995, rs650914, rs650914, rs6505145, rs4509145, rs4509145, rs4509146 , rs7747934, rs4583999, rsl0945364, rsll56143, rsl888349, rsl2353255, rs7027515, rs332185, rs763373, rsl0750861, rsll564173, rs2332631, rsl0399 subset of individuals in the population at risk of sporadic Parkinson's disease even without regard to gender, age or origin. Example 5. First Validation of the selected markers (Spanish Population)
Pacientes y controles  Patients and controls
Casos confirmados de la Enfermedad de Parkinson:  Confirmed cases of Parkinson's disease:
Se reclutaron 372 casos confirmados de PD complejo no consecutivos (es decir que no se recogieron de manera continuada) . La edad media de la serie de pacientes es 71+11,8 años y el rango de edad de 21 a 95 años. El 57,3% de los pacientes (n=213) eran varones y 42,7% (n=159) mujeres. El promedio de la edad de comienzo de la enfermedad de Parkinson de los casos reclutados es de 55,95±12,9 años. El rango de edades de comienzo de la patología oscila entre los 8 y los 83 años. Todos los pacientes son residentes de Cataluña y se reclutaron en el servicio de Neurología del Hospital Clínic de Barcelona entre los años 1997 y 2008. Todos los pacientes estudiados son concordantes con el diagnostico de PD de acuerdo con los criterios diagnósticos comúnmente aceptados para esta enfermedad (21), (37). 372 confirmed cases of non-consecutive complex PD were recruited (that is, they were not collected continuously). The average age of the patient series is 71 + 11.8 years and the age range of 21 to 95 years. 57.3% of the patients (n = 213) were male and 42.7% (n = 159) female. The average age of onset of Parkinson's disease in the recruited cases is 55.95 ± 12.9 years. The age range of onset of the pathology ranges from 8 to 83 years. All patients are residents of Catalonia and were recruited in the Neurology service of the Hospital Clínic de Barcelona between 1997 and 2008. All patients studied are consistent with the diagnosis of PD according to the commonly accepted diagnostic criteria for this disease ( 21), (37).
Los pacientes incluidos han tenido un seguimiento longitudinal en la Unidad de Trastornos del Movimiento del Servicio de Neurología. Del mismo modo su historia familiar ha sido periódicamente actualizada en la misma Unidad. Cuando la historia familiar resultó positiva su grado de certeza se valoró con los criterios de Marder y colaboradores (38). El consentimiento informado se obtuvo de todos los participantes y el comité de ética del Hospital Clínic evaluó y aprobó de forma explícita y específica esta investigación.  The included patients have had a longitudinal follow-up in the Movement Disorders Unit of the Neurology Department. In the same way your family history has been periodically updated in the same Unit. When the family history was positive, its degree of certainty was assessed with the criteria of Marder et al. (38). Informed consent was obtained from all participants and the ethics committee of the Hospital Clínic explicitly and specifically evaluated and approved this research.
Controles : Controls:
Seleccionamos 588 controles de la población general española del Biobanco privado de ADN perteneciente a neoCodex SL (España). La selección fue aleatoria, tan sólo atendiendo a la condición de que el lugar de nacimiento del control y sus ancestros inmediatos fuera España. La edad media de los controles es 47,5+9 años. En el grupo control contabilizamos 213 (37,1%) varones y 351 (62,9%) mujeres.  We selected 588 controls of the general Spanish population of the private DNA Biobank belonging to neoCodex SL (Spain). The selection was random, only considering the condition that the birthplace of the control and its immediate ancestors was Spain. The average age of the controls is 47.5 + 9 years. In the control group we accounted for 213 (37.1%) men and 351 (62.9%) women.
Extracción de ADN Extraction of DNA
El DNA de pacientes y controles se extrajo utilizando la tecnología automatizada MAGNApure de Roche (Suiza) siguiendo las instrucciones del fabricante a partir de 300 ul de Sangre total periférica obtenida mediante venopunción. Una vez finalizado el proceso automático de extracción se procede al control de cantidad y de calidad del material obtenido. Para ello se emplean técnicas nano-espectrofotométricas que permiten medir las absorbancias de la muestra a 260 y 280 nm (Nanodrop, Thermo Scientific) . Con los datos se calculan tanto la concentración de la muestra como el índice 260nm/280nm que indica la pureza del material obtenido. Patient and control DNA was extracted using automated MAGNApure technology from Roche (Switzerland) following the manufacturer's instructions from 300 ul of peripheral total blood obtained by venipuncture. Once the automatic extraction process is finished, proceed to quantity and quality control of the material obtained. For this, nano-spectrophotometric techniques are used to measure the absorbances of the sample at 260 and 280 nm (Nanodrop, Thermo Scientific). With the data both the concentration of the sample and the 260nm / 280nm index that indicates the purity of the material obtained are calculated.
Ejemplo 6. Segunda serie de validación (obtenida de NCBI) Pacientes y controles Example 6. Second validation series (obtained from NCBI) Patients and controls
Pacientes: casos confirmados de la enfermedad de Parkinson: Patients: confirmed cases of Parkinson's disease:
Los datos genotípicos de las muestras utilizadas para la tercera replica (segunda validación) de nuestro estudio se obtuvieron directamente de NCBI (NINDS-Genome-Wide Genotyping in Parkinson' s Disease, http : //www . ncbi . nlm. nih . gov/pro ects/gap/cgibin/ study. cgi?stu dy_id=phs000089. v3. p2 ; Fecha de consulta 31 de Enero de 2010) .  Genotypic data from the samples used for the third replication (second validation) of our study were obtained directly from NCBI (NINDS-Genome-Wide Genotyping in Parkinson's Disease, http: // www. Ncbi. Nlm. Nih. Gov / pro ects / gap / cgibin / study. cgi? stu dy_id = phs000089. v3. p2; Date of consultation January 31, 2010).
En concreto empleamos 661 casos del estudio NINDS (National Institute of Neurological Disorders, NIH, EEUU) que están depositados en las bases de datos de los Laboratorios de Coriell (www.coriell.org; fecha de consulta 31 de Enero de 2010) . Todos los pacientes eran blancos y norte-americanos de ascendencia norte-europea. Los pacientes tenían diagnóstico clínico de Enfermedad de Parkinson idiopática. El promedio de edad de aparición de los síntomas es 55.91 años. Todos los pacientes sufrían bradiquinesia (trastorno caracterizado por el enlentecimiento de todos los movimientos voluntarios y el habla, como la causada por el parkinsonismo, otras alteraciones extrapiramidales y ciertos tranquilizantes) y alguno (o varios) de los siguientes hallazgos: temblor de extremidades, inestabilidad postural y respuesta al tratamiento ant i-parkinsoniano adecuada. Los detalles de estos pacientes están recogidos en el articulo publicado por J. Simón-Sánchez y colaboradores (39). Specifically, we used 661 cases of the NINDS study (National Institute of Neurological Disorders, NIH, USA) that are deposited in the databases of Coriell Laboratories (www.coriell.org; date of consultation January 31, 2010). All patients were white and North American of North European descent. Patients had a clinical diagnosis of idiopathic Parkinson's disease. The average age of onset of symptoms is 55.91 years. All patients suffered from bradykinesia (disorder characterized by the slowing of all voluntary movements and speech, such as that caused by parkinsonism, other extrapyramidal alterations and certain tranquilizers) and some (or several) of the following findings: limb tremor, instability postural and response to adequate anti- parkinsonian treatment. The Details of these patients are included in the article published by J. Simón-Sánchez et al. (39).
Controles :  Controls:
Los 532 controles de esta validación se recogieron de los paneles pre-compilados en los repositorios de Coriell. Todos los individuos seleccionados manifestaron su condición de blancos caucasianos libres de enfermedades neuro- psiquiátricas y se interrogaron acerca de enfermedades neurológicas tales como Enfermedad de Alzheimer, esclerosis lateral amioatrófica, ataxias, autismo, depresión mayor, enfermedades cerebrovasculares , demencia, enfermedad de Parkinson y esquizofrenia. Sus padres ni sus hermanos tampoco presentaban enfermedades neurológicas. La edad media de estos controles es de 58 años (15-98 años).  The 532 controls of this validation were collected from pre-compiled panels in the Coriell repositories. All selected individuals expressed their status as Caucasian targets free of neuropsychiatric diseases and were questioned about neurological diseases such as Alzheimer's disease, amioatrophic lateral sclerosis, ataxias, autism, major depression, cerebrovascular diseases, dementia, Parkinson's disease and schizophrenia . His parents and siblings did not have neurological diseases either. The average age of these controls is 58 years (15-98 years).
Extracción SNPs desde NCBI (National Center for Biotechnological Information) : SNPs extraction from NCBI (National Center for Biotechnological Information):
Los genotipos pertenecientes a estos individuos se extrajeron directamente de las matrices obtenidas de NCBI aplicando tecnologías informáticas convencionales. En concreto obtuvimos los 384 marcadores mencionados en la tabla 2 aplicando sucesivamente a las matrices de NCBI los comandos -proxy-impute all (para inferir los marcadores que no estuvieran presentes) , --snp (para seleccionar de la matriz la lista de SNPs de interés) y -make-bed (para generar una nueva matriz de datos exclusivamente con los marcadores seleccionados) . Genotypes belonging to these individuals were extracted directly from the matrices obtained from NCBI using conventional computer technologies. Specifically, we obtained the 384 markers mentioned in table 2 by successively applying to the NCBI matrices the commands -proxy-impute all (to infer the markers that were not present), --snp (to select from the matrix the list of SNPs from interest) and -make-bed (to generate a new data matrix exclusively with the selected markers).
Cálculos estadísticos: Statistical Calculations:
Igualmente que en el caso anterior, los genotipos crudos obtenidos de la matrices se emplearon como input para el software HFCC y otros programas bioinformáticos , como PLINK, que se utilizan para evaluar los resultados del estudio. PLINK (6), y en concreto el comando -assoc, permite realizar el estudio univariante de cada SNP respecto a la enfermedad. El comando -linear permite el análisis de regresión lineal ajustado. El comando -logistic permite el análisis de regresión logística ajustado por edad y sexo, los comandos - within y -homog permiten el cálculo del índice de heterogeneidad y el estadístico de Breslow-Day y el análisis estratificado por series. El comando --meta-analysis permite el análisis conjunto de todos los estudios. HFCC se emplea para el análisis bi-variante. El plot de Forest se realiza con el software libre episheetAs in the previous case, the raw genotypes obtained from the matrices were used as input for the HFCC software and other bioinformatics programs, such as PLINK, which are used to evaluate the results of the study. PLINK (6), and in particular the -assoc command, allows the univariate study of each SNP with respect to the disease. The -linear command allows the adjusted linear regression analysis. The -logistic command allows the logistic regression analysis adjusted by age and sex, the - within and -homog commands allow the calculation of the heterogeneity index and the Breslow-Day statistic and the stratified analysis by series. The --meta-analysis command allows the joint analysis of all studies. HFCC is used for bi-variant analysis. The plot of Forest is done with the free episheet software
(http://www.drugepi.org/links/downloads/ episheet.xls). El análisis discriminante y los cálculos de área bajo la curva se realizan con SPSS. (http://www.drugepi.org/links/downloads/ episheet.xls). Discriminant analysis and area calculations under the curve are performed with SPSS.
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Claims

REIVINDICACIONES
1. Procedimiento in vitro para determinar la predisposición a padecer la enfermedad de Parkinson en una muestra aislada de un individuo, que comprende el análisis genotipico en dicha muestra del conjunto de SNPs formado por: rs577562, rs2986574, rsllll9097, rs4658673, rs4490160, rsl0496075, rsl2987286, rs847126, rs9036, rs6805546, rs4505714, 1. In vitro procedure for determining the predisposition to suffer from Parkinson's disease in an isolated sample of an individual, which comprises the genotypic analysis in said sample of the set of SNPs formed by: rs577562, rs2986574, rsllll9097, rs4658673, rs4490160, rsl0496075, rsl2987286, rs847126, rs9036, rs6805546, rs4505714,
rs7650598, rsl0155062, rs218280, rs869026, rs3094694, rsl549355, rs7747934, rs4583999, rsl0945364, rsll56143, rsl888349, rsl2353255, rs7027515, rs332185, rs763373, rsl0750861, rsll564173, rs2332631, rsl039189, rs3936139, rs8109493, rs761267, rs6518223, rsl364658, rs353111. rs7650598, rsl0155062, rs218280, rs869026, rs3094694, rsl549355, rs7747934, rs4583999, rsl0945364, rsll56143, rsl888349, rsl2353255, rs7027515, rs332185, rs763373, rsl0750861, rsll564173, rs2332631, rsl039189, rs3936139, rs8109493, rs761267, rs6518223, rsl364658, rs353111.
2. Procedimiento según la reivindicación 1 que comprende el análisis conjunto de los SNPs rs869026 y rsl0945364, situados en los cromosomas 5 y 6, respectivamente. 2. The method according to claim 1 comprising the joint analysis of the SNPs rs869026 and rsl0945364, located on chromosomes 5 and 6, respectively.
3. Conjunto de reactivos o kit para la determinación de la predisposición a padecer la enfermedad de Parkinson en una muestra aislada de un individuo, caracterizado porque 3. Set of reagents or kit for the determination of the predisposition to suffer from Parkinson's disease in an isolated sample of an individual, characterized in that
comprende un conjunto de ácidos nucleicos capaces de hibridar y detecta r el conjunto de SNPs formado por: rs577562, rs2986574, rsllll9097, rs4658673, rs4490160, rsl0496075, rs!2987286, rs847126, rs9036, rs6805546, rs4505714, It comprises a set of nucleic acids capable of hybridizing and detecting r the set of SNPs formed by: rs577562, rs2986574, rsllll9097, rs4658673, rs4490160, rsl0496075, rs! 2987286, rs847126, rs9036, rs6805546, rs4505714, rs4505746
rs7650598, rsl0155062, rs218280, rs869026, rs3094694, rs!549355, rs7747934, rs4583999, rs!0945364, rsl!56143, rsl888349, rsl2353255, rs7027515, rs332185, rs763373, rsl0750861, rsll564173, rs2332631, rsl039189, rs3936139, rs8109493, rs761267( rs6518223, rsl364658, rs353111. rs7650598, rsl0155062, rs218280, rs869026, rs3094694, rs! 549355, rs7747934, rs4583999, rs! 0945364, rsl! 56143, rsl888349, rsl2353255, rs7027515, rs332185, rs763373, rsl0750861, rsll564173, rs2332631, rsl039189, rs3936139, rs8109493, rs761267 ( rs6518223, rs6518223, rs6518223, rs6518223,
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