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• W2093694179 abstract "Human narcolepsy is a hypersomnia that is affected by multiple genetic and environmental factors. One genetic factor strongly associated with narcolepsy is the HLA-DRB1*1501-DQB1*0602 haplotype in the human leukocyte antigen region on chromosome 6, whereas the other genetic factors are not clear. To discover additional candidate regions for susceptibility or resistance to human narcolepsy, we performed a genomewide association study, using 23,244 microsatellite markers. Two rounds of screening with the use of pooled DNAs yielded 96 microsatellite markers (including 16 markers on chromosome 6) with significantly different estimated frequencies in case and control pools. Markers not located on chromosome 6 were evaluated by the individual typing of 95 cases and 95 controls; 30 markers still showed significant associations. A strong association was displayed by a marker on chromosome 21 (21q22.3). The surrounding region was subjected to high-density association mapping with 14 additional microsatellite markers and 74 SNPs. One microsatellite marker (D21S0012m) and two SNPs (rs13048981 and rs13046884) showed strong associations (P<.0005; odds ratios 0.19–0.33). These polymorphisms were in a strong linkage disequilibrium, and no other polymorphism in the region showed a stronger association with narcolepsy. The region contains three predicted genes—NLC1-A, NLC1-B, and NLC1-C—tentatively named “narcolepsy candidate-region 1 genes,” and NLC1-A and NLC1-C were expressed in human hypothalamus. Reporter-gene assays showed that the marker D21S0012m in the promoter region and the SNP rs13046884 in the intron of NLC1-A significantly affected expression levels. Therefore, NLC1-A is considered to be a new resistance gene for human narcolepsy. Human narcolepsy is a hypersomnia that is affected by multiple genetic and environmental factors. One genetic factor strongly associated with narcolepsy is the HLA-DRB1*1501-DQB1*0602 haplotype in the human leukocyte antigen region on chromosome 6, whereas the other genetic factors are not clear. To discover additional candidate regions for susceptibility or resistance to human narcolepsy, we performed a genomewide association study, using 23,244 microsatellite markers. Two rounds of screening with the use of pooled DNAs yielded 96 microsatellite markers (including 16 markers on chromosome 6) with significantly different estimated frequencies in case and control pools. Markers not located on chromosome 6 were evaluated by the individual typing of 95 cases and 95 controls; 30 markers still showed significant associations. A strong association was displayed by a marker on chromosome 21 (21q22.3). The surrounding region was subjected to high-density association mapping with 14 additional microsatellite markers and 74 SNPs. One microsatellite marker (D21S0012m) and two SNPs (rs13048981 and rs13046884) showed strong associations (P<.0005; odds ratios 0.19–0.33). These polymorphisms were in a strong linkage disequilibrium, and no other polymorphism in the region showed a stronger association with narcolepsy. The region contains three predicted genes—NLC1-A, NLC1-B, and NLC1-C—tentatively named “narcolepsy candidate-region 1 genes,” and NLC1-A and NLC1-C were expressed in human hypothalamus. Reporter-gene assays showed that the marker D21S0012m in the promoter region and the SNP rs13046884 in the intron of NLC1-A significantly affected expression levels. Therefore, NLC1-A is considered to be a new resistance gene for human narcolepsy. Narcolepsy (MIM 161400) typically appears, without sexual difference, in early adulthood and affects 0.16%–0.18% of the general population of Japan.1Honda Y Matsuki K Census of narcolepsy, cataplexy and sleep life among teen-agers in Fujisawa city.Sleep Res. 1979; 8: 191Google Scholar, 2Mignot E Genetic and familial aspects of narcolepsy.Neurology. 1998; 50: S16-S22Crossref PubMed Google Scholar The disorder is characterized by excessive daytime sleepiness, cataplexy, and pathological manifestation of rapid eye movement (REM) sleep, including hypnagogic hallucinations, sleep paralysis, or sleep-onset REM sleep. Most cases are sporadic, but the risk of the disorder for first-degree relatives of patients with narcolepsy is 1%–2%, ∼10 times greater than the general risk of developing narcolepsy. Only about a third of MZ twins are concordant for narcolepsy.2Mignot E Genetic and familial aspects of narcolepsy.Neurology. 1998; 50: S16-S22Crossref PubMed Google Scholar Therefore, human narcolepsy is considered to be a multifactorial disorder, involving multiple genetic and environmental factors. A genetic susceptibility factor associated with the disorder has been found in the human leukocyte antigen (HLA) class II region: the HLA-DRB1*1501-DQB1*0602 haplotype (HLA-DRB1 [MIM 142857] and HLA-DQB1 [MIM 604305]). Although almost all Japanese patients with narcolepsy carry this haplotype, ∼10% of the general Japanese population also carries it, suggesting that this haplotype is neither necessary nor sufficient for the development of narcolepsy.1Honda Y Matsuki K Census of narcolepsy, cataplexy and sleep life among teen-agers in Fujisawa city.Sleep Res. 1979; 8: 191Google Scholar, 3Juji T Satake M Honda Y Doi Y HLA antigens in Japanese patients with narcolepsy: all the patients were DR2 positive.Tissue Antigens. 1984; 24: 316-319Crossref PubMed Scopus (302) Google Scholar, 4Matsuki K Juji T Tokunaga K Naohara T Satake M Honda Y Human histocompatibility leukocyte antigen (HLA) haplotype frequencies estimated from the data on HLA class I, II, and III antigens in 111 Japanese narcoleptics.J Clin Invest. 1985; 76: 2078-2083Crossref PubMed Scopus (42) Google Scholar, 5Mignot E Lin L Rogers W Honda Y Qiu X Lin X Okun M Hohjoh H Miki T Hsu S Leffell M Grumet F Fernandez-Vina M Honda M Risch N Complex HLA-DR and -DQ interactions confer risk of narcolepsy-cataplexy in three ethnic groups.Am J Hum Genet. 2001; 68: 686-699Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar This conclusion is also supported by another line of reasoning. The penetrance and population frequency of HLA-DRB1*1501 were estimated with the formula described by Ohashi et al.,8Ohashi J Yamamoto S Tsuchiya N Hatta Y Komata T Matsushita M Tokunaga K Comparison of statistical power between 2×2 allele frequency and allele positivity tables in case-control studies of complex disease genes.Ann Hum Genet. 2001; 65: 197-206Crossref PubMed Scopus (105) Google Scholar based on the prevalence of narcolepsy in the Japanese population (0.16%–0.18%)1Honda Y Matsuki K Census of narcolepsy, cataplexy and sleep life among teen-agers in Fujisawa city.Sleep Res. 1979; 8: 191Google Scholar, 2Mignot E Genetic and familial aspects of narcolepsy.Neurology. 1998; 50: S16-S22Crossref PubMed Google Scholar and the results of a case-control association study of this haplotype.7Hohjoh H Terada N Honda Y Juji T Tokunaga K Negative association of the HLA-DRB1*1502-DQB1*0601 haplotype with human narcolepsy.Immunogenetics. 2001; 52: 299-301Crossref PubMed Scopus (12) Google Scholar On the basis of these values and with the formula described by James9James JW Frequency in relatives for an all-or-none trait.Ann Hum Genet. 1971; 35: 47-49Crossref PubMed Scopus (126) Google Scholar and by Risch,10Risch N Assessing the role of HLA-linked and unlinked determinants of disease.Am J Hum Genet. 1987; 40: 1-14PubMed Google Scholar the expected λs value for HLA of Japanese patients with narcolepsy was calculated to be 5.15, much less than the λs of 12 reported for narcolepsy.6Vyse TJ Todd JA Genetic analysis of autoimmune disease.Cell. 1996; 85: 311-318Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar Therefore, genes other than HLA are also expected to contribute to the disease susceptibility. Several candidate regions11Nakayama J Miura M Honda M Miki T Honda Y Arinami T Linkage of human narcolepsy with HLA association to chromosome 4p13-q21.Genomics. 2000; 65: 84-86Crossref PubMed Scopus (72) Google Scholar, 12Dauvilliers Y Blouin JL Neidhart E Carlander B Eliaou JF Antonarakis SE Billiard M Tafti M A narcolepsy susceptibility locus maps to a 5 Mb region of chromosome 21q.Ann Neurol. 2004; 56: 382-388Crossref PubMed Scopus (52) Google Scholar, 13Wieczorek S Jagiello P Arning L Dahmen N Epplen JT Screening for candidate gene regions in narcolepsy using a microsatellite based approach and pooled DNA.J Mol Med. 2004; 82: 696-705Crossref PubMed Scopus (27) Google Scholar and genes14Aldrich MS Narcolepsy.N Engl J Med. 1990; 323: 389-394Crossref PubMed Scopus (119) Google Scholar, 15Chabas D Taheri S Renier C Mignot E The genetics of narcolepsy.Annu Rev Genomics Hum Genet. 2003; 4: 459-483Crossref PubMed Scopus (101) Google Scholar other than HLA have been investigated for association with human narcolepsy involving cataplexy (narcolepsy-cataplexy) and daytime sleepiness. Nevertheless, replicated associations are few, except for tumor necrosis factor-α (TNFA [MIM 191160]) and TNF-receptor 2 (TNFR2 [MIM 191191]).16Hohjoh H Nakayama T Ohashi J Miyagawa T Tanaka H Akaza T Honda Y Juji T Tokunaga K Significant association of a single nucleotide polymorphism in the tumor necrosis factor-alpha (TNF-α) gene promoter with human narcolepsy.Tissue Antigens. 1999; 54: 138-145Crossref PubMed Scopus (104) Google Scholar, 17Hohjoh H Terada N Kawashima M Honda Y Tokunaga K Significant association of the tumor necrosis factor receptor 2 (TNFR2) gene with human narcolepsy.Tissue Antigens. 2000; 56: 446-448Crossref PubMed Scopus (56) Google Scholar, 18Kawashima M Hohjoh H Terada N Komata T Honda Y Tokunaga K Association studies of the tumor necrosis factor-alpha (TNFA) and its receptor 1 (TNFR1) and 2 (TNFR2) genes with human narcolepsy.Korean J Genet. 2001; 23: 365-370Google Scholar, 19Wieczorek S Dahmen N Jagiello P Epplen JT Gencik M Polymorphisms of the tumor necrosis factor receptors: no association with narcolepsy in German patients.J Mol Med. 2003; 81: 87-90PubMed Google Scholar, 20Wieczorek S Gencik M Rujescu D Tonn P Giegling I Epplen JT Dahmen N TNFA promoter polymorphisms and narcolepsy.Tissue Antigens. 2003; 61: 437-442Crossref PubMed Scopus (42) Google Scholar In autosomal recessive canine models that develop narcolepsy-cataplexy with full penetrance, an insertion in the hypocretin (orexin)–receptor type 2 gene (HCRTR2 [MIM 602393]) was found to be responsible for the disorder,21Lin L Faraco J Li R Kadotani H Rogers W Lin X Qiu X de Jong PJ Nishino S Mignot E The sleep disorder canine narcolepsy is caused by a mutation in the hypocretin (orexin) receptor 2 gene.Cell. 1999; 98: 365-376Abstract Full Text Full Text PDF PubMed Scopus (2218) Google Scholar and preprohypocretin-knockout mice exhibit a phenotype similar to narcolepsy-cataplexy.22Chemelli RM Willie JT Sinton CM Elmquist JK Scammell T Lee C Richardson JA Williams SC Xiong Y Kisanuki Y Fitch TE Nakazato M Hammer RE Saper CB Yanagisawa M Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation.Cell. 1999; 98: 437-451Abstract Full Text Full Text PDF PubMed Scopus (2620) Google Scholar For human narcolepsy, which shows multifactorial inheritance, as described above, the hypocretin concentration in cerebrospinal fluid was reduced or undetectable in sporadic narcolepsy,23Nishino S Ripley B Overeem S Lammers GJ Mignot E Hypocretin (orexin) deficiency in human narcolepsy.Lancet. 2000; 355: 39-40Abstract Full Text Full Text PDF PubMed Scopus (1511) Google Scholar and the number of hypothalamic hypocretin neurons was decreased in postmortem narcoleptic brains.24Peyron C Faraco J Rogers W Ripley B Overeem S Charnay Y Nevsimalova S Aldrich M Reynolds D Albin R Li R Hungs M Pedrazzoli M Padigaru M Kucherlapati M Fan J Maki R Lammers GJ Bouras C Kucherlapati R Nishino S Mignot E A mutation in a case of early onset narcolepsy and a generalized absence of hypocretin peptides in human narcoleptic brains.Nat Med. 2000; 6: 991-997Crossref PubMed Scopus (1773) Google Scholar, 25Thannickal TC Moore RY Nienhuis R Ramanathan L Gulyani S Aldrich M Cornford M Siegel JM Reduced number of hypocretin neurons in human narcolepsy.Neuron. 2000; 27: 469-474Abstract Full Text Full Text PDF PubMed Scopus (1701) Google Scholar Although the preprohypocretin (MIM 602358) and hypocretin-receptor genes have been examined for possible association with human narcolepsy, variants in these genes were not detected in most human patients with narcolepsy.26Sakurai T Moriguchi T Furuya K Kajiwara N Nakamura T Yanagisawa M Goto K Structure and function of human prepro-orexin gene.J Biol Chem. 1999; 274: 17771-17776Crossref PubMed Scopus (173) Google Scholar, 27Hungs M Lin L Okun M Mignot E Polymorphisms in the vicinity of the hypocretin/orexin are not associated with human narcolepsy.Neurology. 2001; 57: 1893-1895Crossref PubMed Scopus (54) Google Scholar, 28Olafsdottir BR Rye DB Scammell TE Matheson JK Stefansson K Gulcher JR Polymorphisms in hypocretin/orexin pathway genes and narcolepsy.Neurology. 2001; 57: 1896-1899Crossref PubMed Scopus (67) Google Scholar Therefore, human narcolepsy cannot be explained by mutations in preprohypocretin and hypocretin-receptor genes. There is evidence for a role of autoantibodies in narcolepsy. Recently, mice were injected with purified immunoglobulin G (IgG) fraction from the serum of nine patients who have narcolepsy-cataplexy with the HLA-DQB1*0602 haplotype. These mice exhibited stresslike behaviors, such as crouching posture and piloerection, and narcoleptic-like behavior, such as brief behavioral pauses lasting from a few seconds to a minute during periods of activity.29Smith AJ Jackson MW Neufing P McEvoy RD Gordon TP A functional autoantibody in narcolepsy.Lancet. 2004; 364: 2122-2124Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar Another group revealed that IgG in the cerebrospinal fluid of HLA-DQB1*0602–positive patients with narcolepsy-cataplexy binds to rat hypothalamic proteins.30Black 3rd, JL Silber MH Krahn LE Avula RK Walker DL Pankratz VS Fredrickson PA Slocumb NL Studies of humoral immunity to preprohypocretin in human leukocyte antigen DQB1*0602-positive narcoleptic subjects with cataplexy.Sleep. 2005; 28: 1191-1192PubMed Google Scholar These two reports suggested that cerebrospinal fluid and serum from patients with narcolepsy contain functional autoantibodies that contribute to the pathogenesis of narcolepsy. However, the pathophysiological mechanism and genetic factors underlying human narcolepsy remain unknown. For this study, we performed a genomewide association study, using 23,244 microsatellite markers for the detection of susceptibility/resistance regions to narcolepsy. This strategy is expected to be effective in the search for candidate regions throughout the whole genome, because of the high detection power of case-control association studies.31Risch N Merikangas K The future of genetic studies of complex human diseases.Science. 1996; 273: 1516-1517Crossref PubMed Scopus (4287) Google Scholar, 32Ohashi J Tokunaga K The power of genome-wide association studies of complex disease genes: statistical limitations of indirect approaches using SNP markers.J Hum Genet. 2001; 46: 478-482Crossref PubMed Scopus (67) Google Scholar Microsatellite markers are abundant and interspersed throughout the human genome. Compared with SNPs, microsatellite markers display higher degrees of polymorphisms: multiple alleles exhibit high levels of heterozygosity, so a smaller number of microsatellite markers may provide a reasonable statistical power in association analyses.33Tamiya G Shinya M Imanishi T Ikuta T Makino S Okamoto K Furugaki K et al.Whole genome association study of rheumatoid arthritis using 27,039 microsatellites.Hum Mol Gen. 2005; 14: 2305-2321Crossref PubMed Scopus (104) Google Scholar, 34Ohashi J Tokunaga K Power of genome-wide linkage disequilibrium testing by using microsatellite markers.J Hum Genet. 2003; 48: 487-491Crossref PubMed Scopus (30) Google Scholar Moreover, to reduce the genotyping cost and labor, genomic DNA samples were pooled33Tamiya G Shinya M Imanishi T Ikuta T Makino S Okamoto K Furugaki K et al.Whole genome association study of rheumatoid arthritis using 27,039 microsatellites.Hum Mol Gen. 2005; 14: 2305-2321Crossref PubMed Scopus (104) Google Scholar, 35Barcellos LF Klitz W Field LL Tobias R Bowcock AM Wilson R Nelson MP Nagatomi J Thomson G Association mapping of disease loci, by use of a pooled DNA genomic screen.Am J Hum Genet. 1997; 61: 734-747Abstract Full Text PDF PubMed Scopus (203) Google Scholar in the first and second screenings. We demonstrated elsewhere that this strategy can detect the known association with the HLA region; using 1,265 microsatellite markers on chromosome 6, we detected strong associations between multiple microsatellite markers in the HLA region and human narcolepsy.36Kawashima M Ikuta T Tamiya G Hohjoh H Honda Y Juji T Tokunaga K Inoko H Genome-wide association study of narcolepsy: initial screening on chromosome 6.in: Dupont J Hansen JA HLA 2004: immunobiology of the human MHC. Proceedings of the 13th International Histocompatibility Workshop and Conference. IHWG Press, Seattle2004Google Scholar Here, we extend the strategy to the other chromosomes, using 21,979 additional microsatellite markers. All patients and unaffected individuals were unrelated Japanese adults living in Tokyo or neighboring areas. Genomic DNAs were obtained from 370 patients given a diagnosis of narcolepsy-cataplexy at the Sleep Disorders Clinic of Seiwa Hospital. All patients with narcolepsy carried the HLA-DRB1*1501-DQB1*0602 haplotype. These 370 genomic DNAs were divided randomly into three sets (the first and second sets with 110 samples each and the third set with the remaining 150 samples). The control group comprised 610 unrelated unaffected individuals and an additional 125 individuals positive for HLA-DRB1*1501. The 610 control samples were also divided into three sets (210 samples each in the first and second sets and the remaining 190 samples in the third set). Genomic DNAs were purified from peripheral blood, with the use of a commercial kit (QIAamp Blood Kit [Qiagen]). This study was approved by the research ethics review committees of the University of Tokyo and the Neuropsychiatric Research Institute, which runs Seiwa Hospital. Genomic DNA concentration was measured in triplicate, in accordance with the methods of Collins et al.,37Collins HE Li H Inda SE Anderson J Laiho K Tuomilehto J Seldin MF A simple and accurate method for determination of microsatellite total allele content differences between DNA pools.Hum Genet. 2000; 106: 218-226Crossref PubMed Scopus (65) Google Scholar with the use of a double-stranded DNA quantification kit (PicoGreen [Molecular Probes]) with a microtiter plate reader (SF600 Corona Electric). Genomic DNAs were adjusted to 8 ng/μl. DNAs from 110 patients with narcolepsy and from 210 controls were then mixed, for the first set of case and control pools, named “case-1” and “control-1,” respectively.38Kirov G Williams N Sham P Craddock N Owen MJ Pooled genotyping of microsatellite markers in parent-offspring trios.Genome Res. 2000; 10: 105-115PubMed Google Scholar The second set of pooled DNA (case-2 and control-2) was also prepared from another 110 cases and 210 controls. All microsatellite markers and the methods for microsatellite analysis used in this study are described by Tamiya et al.33Tamiya G Shinya M Imanishi T Ikuta T Makino S Okamoto K Furugaki K et al.Whole genome association study of rheumatoid arthritis using 27,039 microsatellites.Hum Mol Gen. 2005; 14: 2305-2321Crossref PubMed Scopus (104) Google Scholar In brief, PCR primers were designed for amplifying fragments that include the microsatellite polymorphisms. All PCR primers were designed to have an annealing temperature of 57°C. Forward primers were labeled at the 5′ end with fluorescent reagent (6-FAM or HEX [Applied Biosystems]). PCR on pooled DNAs was performed in 20-μl reactions containing 48 ng of pooled DNA, 0.5 units of DNA polymerase (AmpliTaq [Applied Biosystems]), 1× reaction buffer with 1.5 mM MgCl2 provided by the manufacturer (Applied Biosystems), 5 μM of each primer, and 0.25 mM of each deoxyribonucleotide triphosphate (dNTP) in 96- or 384-well plates. The amplification condition consisted of initial denaturation at 96°C for 5 min (hot start), annealing at 57°C for 1 min, and extension at 72°C for 1 min, followed by 40 cycles of denaturation at 96°C for 45 s, annealing at 57°C for 45 s, and extension at 72°C for 1 min, with use of a thermal cycler (GeneAmp PCR system 9700 [Applied Biosystems]). For microsatellite typing of individual samples, PCR was performed in 12-μl reactions containing 2 ng of genomic DNA, 0.25 units of DNA polymerase (AmpliTaq Gold [Applied Biosystems]), 1× reaction buffer with 1.5 mM MgCl2 provided by the manufacturer, 5 μM of each primer, and 0.2 mM of each dNTP in 96- or 384-well plates. The amplification conditions were essentially the same as described above. The PCR products were denatured in formamide (Hi-Di [Applied Biosystems]) at 95°C for 3 min and were separated by electrophoresis, with the use of an automated DNA sequencer with size standards (ABI Prism 3700 Genetic Analyzer, ROX size standard [Applied Biosystems]). The fragment size and the electrophoretograms were analyzed by GeneScan and Genotyper software (Applied Biosystems). To obtain additional microsatellite markers in the narcolepsy candidate–region 1 (NLC1), the sequence of the candidate region was obtained from the University of California–Santa Cruz (UCSC) Genome Browser database (November 2002 version, based on NCBI Build 31). Then, the sequence was searched for repeated elements with the RepeatMasker program. Dinucleotide repeats with repeat number >12, trinucleotide repeats >8, and tetranucleotide to hexanucleotide repeats >5 were chosen. PCR primers were designed as described above, and we evaluated the polymorphism of each microsatellite with pooled DNA, searching for multiple peaks in the electrophoresis. SNPs within the candidate region were selected from the Celera database at average intervals of ∼5 kb, and specific PCR primers were designed. To confirm the polymorphisms of these SNP sites in the Japanese population, we examined 16 samples from patients with narcolepsy by direct sequencing, using a PCR cycle-sequencing kit and an automated DNA sequencer (BigDye Terminator v.3.1 Cycle Sequencing Kit and ABI PRISM 3730 DNA sequencer [Applied Biosystems]). The association analyses with these polymorphic sites were performed by direct sequencing of case and control samples. The sequence of the entire region of NLC1-A, NLC1-B, and NLC1-C genes (based on NCBI Build 35 chromosome 21: 45234058–45250151) was also screened for polymorphisms with 16 samples, and polymorphic sites were subjected to association analyses by direct sequencing of case and control samples. Newly detected polymorphisms have been registered in the dbSNP database. The expression of the predicted genes in candidate region NLC1 was examined by RT-PCR, with the use of poly(A)+ RNA from the human brain, hypothalamus, peripheral blood, sperm, and several organs (i.e., heart, liver, spleen, pancreas, lung, kidney, and skeletal muscle [Bio Chain]). To discriminate PCR products derived from reverse-transcribed mRNA from those derived from genomic DNA, we designed specific forward and reverse primers in the predicted exon 1 and 2 regions, respectively. The primer sets for the predicted genes were as follows: 5′-CTAGGAGGGGAAACTGAGTCC-3′ and 5′-CAGCACAGTTGGAGACATCACT-3′ for NLC1-A, 5′-CCTCACAGCATCCCACATT-3′ and 5′-TTTCTGGAAACAGCCAGGAG-3′ for NLC1-B, and 5′-GCTGAACTGCCTGGACTTTC-3′ and 5′-ACATGTGCTCCCCACCTAAG-3′ for NLC1-C. The thermal cycling profile consisted of initial denaturation at 96°C for 10 min, followed by 35 cycles of denaturation at 96°C for 45 s, annealing at 57°C for 45 s, extension at 72°C for 1 min, and a final extension at 72°C for 5 min, with the use of AmpliTaq Gold polymerase (Applied Biosystems). The PCR products were separated by electrophoresis on 2% agarose gels and were stained with ethidium bromide. The sequences of the amplified products were confirmed by direct sequencing. Reporter-gene assays were performed using constructs containing microsatellite marker D21S0012m and SNP rs13046884 alleles. For D21S0012m, genomic DNAs were obtained from four homozygotes for alleles with AC repeat numbers 8, 9, 10, and 12. A 908-bp fragment within the promoter region including D21S0012m was amplified by PCR, with use of the specific primers 5′-CAAAGGTACCTCCAGTCCACACCCACC-3′ and 5′-GTTTGAGCTCTTTGGCCTGTCCATCAG-3′. Genomic DNA for SNP rs13046884 alleles was obtained from one rs13046884 heterozygote. A 297-bp fragment within NLC1-A intron 1, which includes rs13046884, was amplified using primers 5′-CAAAGGTACCAGGGTTGGACTCCAAAGGGA-3′ and 5′-GTTTGAGCTCGGGTGACTTCTTCACACCCA-3′. PCR was performed (TaKaRa LA Taq [TaKaRa]) with the following thermal cycling profile: denaturation at 96°C for 5 min, followed by 35 cycles at 96°C for 30 s, 60°C for 30 s, and 72°C for 1.5 min. PCR products were digested with SacI and KpnI restriction endonucleases and then were inserted upstream of a firefly luciferase gene in the pGL3-control vector (Promega), with the use of T4 DNA ligase (TaKaRa). Inserted sequences were confirmed by direct sequencing with primers specific to the pGL3-control vector (5′-CATACGCTCTCCATCAAAACAA-3′ and 5′-AAGCCTCCTCACTACTTCTGGA-3′). The neuroblastoma cell line NB-1 and HeLa cells were maintained in accordance with published recommendations (Human Science Research Resources Bank). Then, 0.2 μg of each construct was introduced into the cells by a lipofection method (Effectene Transfection Reagent [Qiagen]), along with 0.02 μg of pRL-SV40 (Promega) as an internal control. Luciferase levels were determined using the DUAL-Luciferase Reporter Assay System (Promega), and firefly luciferase levels were normalized to the levels of renilla luciferase from pRL-SV40. Disease associations with polymorphisms were assessed by Fisher's exact test, with the use of 2×2 contingency tables for each allele. The smallest P value for each marker was selected. Allele frequencies in pooled-DNA typing were estimated from the height of peaks: each allele frequency was determined by dividing the height of each allele by the summed height of all alleles. In individual typing, the significance was evaluated by Fisher's exact test, with the use of 2×m (where m is the number of alleles) and 2×2 contingency tables. The significance level was set at .05 throughout this study, except for homogeneity among samples used in the first, second, and third set, which was tested by means of the Q statistic and was considered significant for P<.10.39Lau J Ioannidis JP Schmid CH Quantitative synthesis in systematic reviews.Ann Intern Med. 1997; 127: 820-826Crossref PubMed Scopus (2155) Google Scholar To assess the extent of pairwise linkage disequilibrium (LD) between polymorphisms, Lewontin's40Lewontin RC The interaction of selection and linkage. II. Optimum models.Genetics. 1964; 50: 757-782PubMed Google ScholarD′ and r2 were calculated using a commercial software package (SNPAlyze-3.2 pro [Dynacom]) based on the expectation-maximation algorithm. D′ and r2 were calculated only for polymorphisms with a minor-allele frequency (MAF) >6%. Pairwise D′ and r2 were plotted at the Cartesian coordinate corresponding to the polymorphism location on the physical map with the use of the GOLD program, as described by Abecasis and Cookson.41Abecasis GR Cookson WO GOLD: graphical overview of linkage disequilibrium.Bioinformatics. 2000; 16: 182-183Crossref PubMed Scopus (677) Google Scholar The 23,244 microsatellite markers used in the genomewide association study are summarized in table 1. To reduce the cost and the technical burden of genomewide association analysis, the DNA-pooling method was applied in the first and second screenings (fig. 1A). Allele frequencies were estimated from the height of individual peaks. To avoid false-negative associations, we performed no correction for multiple comparisons. Figure 1B shows the results of the association analyses in the first screening; the results for the 1,265 microsatellite markers on chromosome 6, which includes the HLA region, were described elsewhere.36Kawashima M Ikuta T Tamiya G Hohjoh H Honda Y Juji T Tokunaga K Inoko H Genome-wide association study of narcolepsy: initial screening on chromosome 6.in: Dupont J Hansen JA HLA 2004: immunobiology of the human MHC. Proceedings of the 13th International Histocompatibility Workshop and Conference. IHWG Press, Seattle2004Google Scholar A total of 2,686 markers (202 of which were on chromosome 6) showed significantly different frequencies between cases and controls. These 2,686 markers were further analyzed in the second screening with pooled DNA samples from different sets of cases and controls, and 96 markers (16 on chromosome 6) remained significantly different between cases and controls and had similar peak patterns between first and second case pools and between first and second control pools.Table 1Numbers and Mean Intervals of Microsatellite Markers on Each ChromosomeChromosomeMarker NumberMean Interval (kb/marker)11,949118.722,100109.131,745110.441,466118.851,467116.861,265125.771,430110.581,132118.19971120.4101,145117.5111,124124.3121,002127.913775125.914663132.715565142.816614124.417621131.018634126.119451123.320490122.821284122.922255133.3X992137.4Y104287.0Note.—The total number of microsatellite markers used in the first and second screenings was 23,244. The mean interval of these markers throughout the genome was 130.3 kb, the mean heterozygosity was 0.67, and the mean allele number was 6.4. O" @default.
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• W2093694179 date "2006-08-01" @default.
• W2093694179 modified "2023-10-17" @default.
• W2093694179 title "Genomewide Association Analysis of Human Narcolepsy and a New Resistance Gene" @default.
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