Common Variants in the BMP2, BMP4, and HJV Genes of the Hepcidin Regulation Pathway Modulate HFE Hemochromatosis Penetrance
2007; Elsevier BV; Volume: 81; Issue: 4 Linguagem: Inglês
10.1086/520001
ISSN1537-6605
AutoresJacqueline Milet, Valérie Déhais, Catherine Bourgain, Anne Marie Jouanolle, Annick Mosser, M. Perrin, Jeff Morçet, Pierre Brissot, Véronique David, Yves Deugnier, Jean Mosser,
Tópico(s)Trace Elements in Health
ResumoMost cases of genetic hemochromatosis (GH) are associated with the HFE C282Y/C282Y (p.Cys282Tyr/p.Cys282Tyr) genotype in white populations. The symptoms expressed by C282Y homozygotes are extremely variable. Only a few suffer from an overt disease. Several studies have suggested that, in addition to environmental factors, a genetic component could explain a substantial part of this phenotypic variation, although very few genetic factors have been identified so far. In the present study, we tested the association between common variants in candidate genes and hemochromatosis penetrance, in a large sample of C282Y homozygotes, using pretherapeutic serum ferritin level as marker of hemochromatosis penetrance. We focused on two biologically relevant gene categories: genes involved in non-HFE GH (TFR2, HAMP, and SLC40A1) and genes involved in the regulation of hepcidin expression, including genes from the bone morphogenetic protein (BMP) regulatory pathway (BMP2, BMP4, HJV, SMAD1, SMAD4, and SMAD5) and the IL6 gene from the inflammation-mediated regulation pathway. A significant association was detected between serum ferritin level and rs235756, a common single-nucleotide polymorphism (SNP) in the BMP2 genic region (P=4.42×10−5). Mean ferritin level, adjusted for age and sex, is 655 ng/ml among TT genotypes, 516 ng/ml in TC genotypes, and 349 ng/ml in CC genotypes. Our results further suggest an interactive effect on serum ferritin level of rs235756 in BMP2 and a SNP in HJV, with a small additive effect of a SNP in BMP4. This first reported association between common variants in the BMP pathway and iron burden suggests that full expression of HFE hemochromatosis is linked to abnormal liver expression of hepcidin, not only through impairment in the HFE function but also through functional modulation in the BMP pathway. Our results also highlight the BMP regulation pathway as a good candidate for identification of new modifier genes. Most cases of genetic hemochromatosis (GH) are associated with the HFE C282Y/C282Y (p.Cys282Tyr/p.Cys282Tyr) genotype in white populations. The symptoms expressed by C282Y homozygotes are extremely variable. Only a few suffer from an overt disease. Several studies have suggested that, in addition to environmental factors, a genetic component could explain a substantial part of this phenotypic variation, although very few genetic factors have been identified so far. In the present study, we tested the association between common variants in candidate genes and hemochromatosis penetrance, in a large sample of C282Y homozygotes, using pretherapeutic serum ferritin level as marker of hemochromatosis penetrance. We focused on two biologically relevant gene categories: genes involved in non-HFE GH (TFR2, HAMP, and SLC40A1) and genes involved in the regulation of hepcidin expression, including genes from the bone morphogenetic protein (BMP) regulatory pathway (BMP2, BMP4, HJV, SMAD1, SMAD4, and SMAD5) and the IL6 gene from the inflammation-mediated regulation pathway. A significant association was detected between serum ferritin level and rs235756, a common single-nucleotide polymorphism (SNP) in the BMP2 genic region (P=4.42×10−5). Mean ferritin level, adjusted for age and sex, is 655 ng/ml among TT genotypes, 516 ng/ml in TC genotypes, and 349 ng/ml in CC genotypes. Our results further suggest an interactive effect on serum ferritin level of rs235756 in BMP2 and a SNP in HJV, with a small additive effect of a SNP in BMP4. This first reported association between common variants in the BMP pathway and iron burden suggests that full expression of HFE hemochromatosis is linked to abnormal liver expression of hepcidin, not only through impairment in the HFE function but also through functional modulation in the BMP pathway. Our results also highlight the BMP regulation pathway as a good candidate for identification of new modifier genes. Genetic hemochromatosis (GH) is a group of hereditary disorders proceeding from an impairment in the production of the key regulator of plasma iron, hepcidin,1Loréal O Haziza-Pigeon C Troadec MB Detivaud L Turlin B Courselaud B Ilyin G Brissot P Hepcidin in iron metabolism.Curr Protein Pept Sci. 2005; 6: 279-291Crossref PubMed Scopus (41) Google Scholar and resulting in progressive iron loading of parenchymas. Four recessive forms of GH are currently described.2Brissot P de Bels F Current approaches to the management of hemochromatosis. American Society of Hematology, Washington, DC2006: 36-41Google Scholar Two late-onset, adult forms (HFE [MIM 235200] and HFE3 [MIM 604250]) are related to mutations in HFE (MIM 235200) or TFR2 (MIM 604720), the gene encoding the receptor transferring 2 protein. Two early-onset, juvenile forms (JH [MIM 602390]) are secondary to mutations in either the gene encoding hemojuvelin (HJV [MIM 608374]) or the gene encoding hepcidin (HAMP [MIM 606464]). The "ferroportin disease" (HFE4 [MIM 606069]), related to mutation in the SLC40A1 gene (SLC40A1 [MIM 604653]) coding for the metal-transporter ferroportin regulated by hepcidin, is a closely related but not classic form of GH, because of a dominant transmission usually with predominant mesenchymal iron deposition.3Pietrangelo A Non-HFE hemochromatosis.Semin Liver Dis. 2005; 25: 450-460Crossref PubMed Scopus (63) Google Scholar HFE hemochromatosis accounts for >95% of GH cases in white populations.4Brissot P Moirand R Jouanolle AM Guyader D Le Gall JY Deugnier Y David V A genotypic study of 217 unrelated probands diagnosed as "genetic hemochromatosis" on "classical" phenotypic criteria.J Hepatol. 1999; 30: 588-593Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar It is related to one major mutation, C282Y, with a reported frequency of 5%–15%.5Burt MJ George PM Upton JD Collett JA Frampton CM Chapman TM Walmsley TA Chapman BA The significance of haemochromatosis gene mutations in the general population: implications for screening.Gut. 1998; 43: 830-836Crossref PubMed Scopus (203) Google Scholar, 6Jouanolle AM Fergelot P Raoul ML Gandon G Roussey M Deugnier Y Feingold J Le Gall JY David V Prevalence of the C282Y mutation in Brittany: penetrance of genetic hemochromatosis?.Ann Genet. 1998; 41: 195-198PubMed Google Scholar, 7Merryweather-Clarke AT Simonsen H Shearman JD Pointon JJ Norgaard-Pedersen B Robson KJ A retrospective anonymous pilot study in screening newborns for HFE mutations in Scandinavian populations.Hum Mutat. 1999; 13: 154-159Crossref PubMed Scopus (52) Google Scholar The biochemical or clinical symptoms expressed by C282Y homozygotes are extremely variable.8Moirand R Jouanolle AM Brissot P Le Gall JY David V Deugnier Y Phenotypic expression of HFE mutations: a French study of 1110 unrelated iron-overloaded patients and relatives.Gastroenterology. 1999; 116: 372-377Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar Only a few suffer from an overt disease consisting of various associated symptoms, including osteoarticular damage, cirrhosis, diabetes, hypogonadism, arrhythmia, and heart failure. Most C282Y homozygotes display a mild disease limited to biochemical abnormalities (increased transferrin saturation, with or without elevated serum ferritin), with either absent or mild clinical symptoms. Moreover, some homozygotes may show no expression throughout their lives.9Coppin H Bensaid M Fruchon S Borot N Blanche H Roth MP Longevity and carrying the C282Y mutation for haemochromatosis on the HFE gene: case control study of 492 French centenarians.BMJ. 2003; 327: 132-133Crossref PubMed Scopus (39) Google Scholar As a whole, estimates of the penetrance of C282Y homozygosity by studies have ranged from 1% to 90%.5Burt MJ George PM Upton JD Collett JA Frampton CM Chapman TM Walmsley TA Chapman BA The significance of haemochromatosis gene mutations in the general population: implications for screening.Gut. 1998; 43: 830-836Crossref PubMed Scopus (203) Google Scholar, 10Adams PC Reboussin DM Barton JC McLaren CE Eckfeldt JH McLaren GD Dawkins FW Acton RT Harris EL Gordeuk VR et al.Hemochromatosis and iron-overload screening in a racially diverse population.N Engl J Med. 2005; 352: 1769-1778Crossref PubMed Scopus (534) Google Scholar, 11Beutler E Felitti VJ Koziol JA Ho NJ Gelbart T Penetrance of 845G→A (C282Y) HFE hereditary haemochromatosis mutation in the USA.Lancet. 2002; 359: 211-218Abstract Full Text Full Text PDF PubMed Scopus (704) Google Scholar, 12Deugnier Y Jouanolle AM Chaperon J Moirand R Pithois C Meyer JF Pouchard M Lafraise B Brigand A Caserio-Schoenemann C et al.Gender-specific phenotypic expression and screening strategies in C282Y-linked haemochromatosis: a study of 9396 French people.Br J Haematol. 2002; 118: 1170-1178Crossref PubMed Scopus (73) Google Scholar, 13Olynyk JK Cullen DJ Aquilia S Rossi E Summerville L Powell LW A population-based study of the clinical expression of the hemochromatosis gene.N Engl J Med. 1999; 341: 718-724Crossref PubMed Scopus (627) Google Scholar This wide range reflects the variability in the definition of disease penetrance, with some studies referring to the level of iron burden (serum ferritin, liver iron concentration…) using disparate thresholds, others to organ damage, and most to mixed criteria. These results are complicated by the absence of a strict correlation between the level of iron burden and organ-damage expression. However, they strongly support the involvement of factors modulating disease expressivity. With respect to iron loading, environmental factors have been poorly investigated in humans, although it is likely that some may be relevant, such as alimentary regimen and blood donation.11Beutler E Felitti VJ Koziol JA Ho NJ Gelbart T Penetrance of 845G→A (C282Y) HFE hereditary haemochromatosis mutation in the USA.Lancet. 2002; 359: 211-218Abstract Full Text Full Text PDF PubMed Scopus (704) Google Scholar Several results also suggest the role of additional genetic factors. The incidence of GH-related conditions is higher in relatives of clinically affected probands than in relatives of probands with only elevated transferrin saturation,14Bulaj ZJ Ajioka RS Phillips JD LaSalle BA Jorde LB Griffen LM Edwards CQ Kushner JP Disease-related conditions in relatives of patients with hemochromatosis.N Engl J Med. 2000; 343: 1529-1535Crossref PubMed Scopus (175) Google Scholar and concordance of iron store indices in GH-affected families between same-sex siblings homozygous C282Y is high.15Whiting PW Fletcher LM Dixon JK Gochee P Powell LW Crawford DH Concordance of iron indices in homozygote and heterozygote sibling pairs in hemochromatosis families: implications for family screening.J Hepatol. 2002; 37: 309-314Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar On the basis of a twin study, Whitfield et al.16Whitfield JB Cullen LM Jazwinska EC Powell LW Heath AC Zhu G Duffy DL Martin NG Effects of HFE C282Y and H63D polymorphisms and polygenic background on iron stores in a large community sample of twins.Am J Hum Genet. 2000; 66: 1246-1258Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar estimated that HFE explains only part of the genetic component of iron-store variation. In mice, differences in hepatic iron loading in two Hfe-deficient mice strains have been associated with a polygenic pattern of inheritance.17Bensaid M Fruchon S Mazeres C Bahram S Roth MP Coppin H Multigenic control of hepatic iron loading in a murine model of hemochromatosis.Gastroenterology. 2004; 126: 1400-1408Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar Apart from sex, only a few genetic factors have been identified as putatively modifying disease expression. A mitochondrial polymorphism was reported as more frequent in C282Y homozygotes with hemochromatosis than in nonexpressing C282Y homozygotes.18Livesey KJ Wimhurst VL Carter K Worwood M Cadet E Rochette J Roberts AG Pointon JJ Merryweather-Clarke AT Bassett ML et al.The 16189 variant of mitochondrial DNA occurs more frequently in C282Y homozygotes with haemochromatosis than those without iron loading.J Med Genet. 2004; 41: 6-10Crossref PubMed Scopus (29) Google Scholar However, in another study, no association has been observed between the ferritin level of C282Y homozygotes and the same polymorphism.19Beutler E Beutler L Lee PL Barton JC The mitochondrial nt 16189 polymorphism and hereditary hemochromatosis.Blood Cells Mol Dis. 2004; 33: 344-345Crossref PubMed Scopus (9) Google Scholar Mutations in two other genes involved in iron metabolism, HJV and HAMP, have been clearly associated with higher iron indices in a French cohort of C282Y homozygotes,20Le Gac G Scotet V Ka C Gourlaouen I Bryckaert L Jacolot S Mura C Ferec C The recently identified type 2A juvenile haemochromatosis gene (HJV), a second candidate modifier of the C282Y homozygous phenotype.Hum Mol Genet. 2004; 13: 1913-1918Crossref PubMed Scopus (97) Google Scholar, 21Merryweather-Clarke AT Cadet E Bomford A Capron D Viprakasit V Miller A McHugh PJ Chapman RW Pointon JJ Wimhurst VL et al.Digenic inheritance of mutations in HAMP and HFE results in different types of haemochromatosis.Hum Mol Genet. 2003; 12: 2241-2247Crossref PubMed Scopus (222) Google Scholar, 22Jacolot S Le Gac G Scotet V Quere I Mura C Ferec C HAMP as a modifier gene that increases the phenotypic expression of the HFE pC282Y homozygous genotype.Blood. 2004; 103: 2835-2840Crossref PubMed Scopus (141) Google Scholar but the mutations identified are rare, with a frequency of heterozygous carriers <2% among C282Y homozygotes. Hepcidin is a peptide hormone produced by the liver that controls plasma iron concentration and iron-tissue distribution by inhibiting intestinal iron absorption, iron recycling by macrophages, and iron mobilization from the liver. Interestingly, hepcidin levels are abnormally low in patients with adult or juvenile forms of GH. Babitt et al.23Babitt JL Huang FW Wrighting DM Xia Y Sidis Y Samad TA Campagna JA Chung RT Schneyer AL Woolf CJ et al.Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression.Nat Genet. 2006; 38: 531-539Crossref PubMed Scopus (792) Google Scholar recently demonstrated that hepcidin expression is induced by the bone morphogenetic protein (BMP)–signaling pathway. This pathway involves BMP2 and BMP4 proteins and the BMP coreceptor HJV, which phosphorylates receptor-activated Smad proteins (R-Smads) 1 and 5. Phosphorylated R-Smads form a complex with the common mediator Smad4 (Co-Smad), which allows signal transduction into the nucleus for hepcidin gene induction. The aim of the present study was to search for relatively frequent variants (as opposed to the rare mutations already known) in genes that modify the serum ferritin levels in C282Y homozygotes. Given these recent molecular data, we focus on variants in two pathophysiologically relevant gene categories: genes involved in non–HFE hemochromatosis and genes involved in the regulation of hepcidin expression. In a large sample of C282Y homozygotes, we tested for association between initial serum ferritin levels and SNPs located within or near 10 genes: 3 non–HFE-GH genes (TFR2, HAMP, and SLC40A1), 6 BMP signaling pathway genes (BMP2 [MIM 112261], BMP4 [MIM 112262], HJV, SMAD1 [MIM 601595], SMAD4 [MIM 600993], and SMAD5 [MIM 603110]), and the IL6 gene (IL6 [MIM 147620]) coding for an inflammatory cytokine known to increase inflammation-mediated hepcidin expression. We detected a significant association between a SNP in the BMP2 genic region and serum ferritin level, adjusted for age and sex. Given the biologically relevant gene interactions along the BMP regulatory pathway, our data suggest an additive effect on serum ferritin level of the BMP2 SNP and of a SNP in BMP4, with an interaction effect between the BMP2 SNP and a SNP in HJV. The database of the Family Screening Centre for Hemochromatosis8Moirand R Jouanolle AM Brissot P Le Gall JY David V Deugnier Y Phenotypic expression of HFE mutations: a French study of 1110 unrelated iron-overloaded patients and relatives.Gastroenterology. 1999; 116: 372-377Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar comprises all C282Y homozygous probands referred to the Liver Unit in Rennes, France, since 1990 and their relatives who received diagnoses through a systematic family-screening policy. At the time of the study, 1,319 C282Y homozygotes were recorded, among whom 729 unrelated probands fulfilled inclusion criteria: availability of (i) sex, (ii) age at diagnosis, (iii) serum ferritin level at diagnosis before any venesection therapy, and (iv) DNA sample stored at −20°C. Of these 729 subjects, 592 gave their written informed consent to participate in the study, in accordance with the protocol validated by the committee of ethics of Rennes on November 10, 2004. Of these 592 subjects, 262 were women and 330 were men. Mean (SD) age was 46 (14) years for women and 44 (13) years for men. Median value (25th–75th percentile range) for ferritin levels was 1,040.5 ng/ml (582–2,356) in men and 400.5 ng/ml (186–699) in women. Ferritin data were normalized using a loge transformation. Log-transformed ferritin levels were adjusted for age, with consideration of age groups of 10 years, and sex. The final multiple-regression model includes a parameter for each of the seven independent age groups, a parameter for sex, and an interaction parameter, Age × Sex, for each age group. Genomic DNA was extracted from peripheral blood cells by the phenol-chloroform method or by use of the Flexigen DNA kit (Qiagen). SNPs in the 10 candidate genes were selected from the CEU HapMap database. For each gene, a region including the complete genic sequence and the upstream and downstream intergenic sequences was delimited. The set of tag SNPs was identified for each region, so that all the SNPs with a minor-allele frequency (MAF) ⩾5% in the database have a pairwise r2⩾0.8 with at least one tag SNP. Tagging was performed using the algorithm implemented in Tagger.24de Bakker PI Yelensky R Pe'er I Gabriel SB Daly MJ Altshuler D Efficiency and power in genetic association studies.Nat Genet. 2005; 37: 1217-1223Crossref PubMed Scopus (1455) Google Scholar Two coding SNPs located within the IL6 gene were added to the list. A total of 81 SNPs were included in the study. SNP genotyping followed Custom SNP Genotyping Assays consisting of a mix of unlabeled PCR primers and TaqMan minor groove binder (MGB) SNP-allele-specific probe. PCR primers and probes used for allelic discrimination were designed and purchased from the Applied Biosystems "assay on demand" or "assay by design." Genotyping followed the Applied Biosystems protocol. Briefly, PCR was performed in a final volume of 5 μl containing 10 ng of sample DNA, 0.625 μl of custom SNP-specific Assay Mix, and 2.5 μl of Universal Master Mix no AmpErase UNG. Amplification was allowed to proceed for 40 cycles of 15 s at 95°C and 60 s at 60°C. Automatic genotype call was performed <24 h after PCR, by scanning microtitration plates on the 7900HT Fast Real-Time PCR, which provides the SDS2.3 software (Applied Biosystems). Assays for rs1880241 (in IL6) and rs6596286 (in SMAD5) were unsuccessful. Of the 79 SNPs genotyped, 4 SNPs (rs1005464 in BMP2, rs10498466 and rs4901473 in BMP4, and rs3764942 in SMAD5) were not in Hardy-Weinberg equilibrium and therefore were excluded from the analyses. Mean genotyping success rate for the 75 remaining SNPs was 99.1%. Correlation between allele frequencies of the 75 SNPs in our sample and allele frequencies in the CEU HapMap data was very high (regression r2=0.94). The linkage disequilibrium (LD) structure among SNPs was examined with Haploview.25Barrett JC Fry B Maller J Daly MJ Haploview: analysis and visualization of LD and haplotype maps.Bioinformatics. 2005; 21: 263-265Crossref PubMed Scopus (11431) Google Scholar The mean r2 between our markers, computed on the whole sample, was 0.10. All subsequent statistical analyses were performed using R.26Team RDC R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna2005Google Scholar A linear regression was used to test for association between each individual SNP and ferritin. Both allelic and genotypic associations were considered. The sensitivity of association results to the inclusion of phenotypic outliers was evaluated. Because the results were fully concordant, we present and discuss only results based on the whole sample. To correct for multiple testing, the effective number of independent tests was assessed using the method of Li and Ji27Li J Ji L Adjusting multiple testing in multilocus analyses using the eigenvalues of a correlation matrix.Heredity. 2005; 95: 221-227Crossref PubMed Scopus (888) Google Scholar as implemented in the SNPSpd software.28Nyholt DR A simple correction for multiple testing for single-nucleotide polymorphisms in linkage disequilibrium with each other.Am J Hum Genet. 2004; 74: 765-769Abstract Full Text Full Text PDF PubMed Scopus (1314) Google Scholar Following this procedure, our set of 75 SNPs is equivalent to 62 independent tests. When a Bonferroni correction is applied, an individual significance threshold of 8×10−4 should be used to control a global 5% type I error. Association between haplotypes and ferritin was tested using the efficient score statistic proposed by Schaid et al.29Schaid DJ Rowland CM Tines DE Jacobson RM Poland GA Score tests for association between traits and haplotypes when linkage phase is ambiguous.Am J Hum Genet. 2002; 70: 425-434Abstract Full Text Full Text PDF PubMed Scopus (1539) Google Scholar and implemented in haplo.stats R-Package v1.2.2 (Schaid Lab Web site) with a permutation-based assessment of the P values. We favored this relatively simple regression-based approach over more sophisticated ones based on population history modeling,30Morris AP Whittaker JC Balding DJ Little loss of information due to unknown phase for fine-scale linkage-disequilibrium mapping with single-nucleotide-polymorphism genotype data.Am J Hum Genet. 2004; 74: 945-953Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar because our sample is a subsample selected from the general population with an unknown selection scheme. In the absence of a clear evaluation of the consequences of such a selection scheme on sophisticated haplotype tests, we favored a robust approach.31Schaid DJ Evaluating associations of haplotypes with traits.Genet Epidemiol. 2004; 27: 348-364Crossref PubMed Scopus (251) Google Scholar Haplotypes comprising all SNPs were considered for genes with <10 SNPs. Otherwise, haplotypes comprising five SNPs were considered, with a sliding window of one SNP to browse the gene. Sensitivity to window size was evaluated. Because results were fully concordant, we present and discuss only results based on five-SNP windows. Association of SNP combinations within genes was further analyzed using unphased, multimarker data. A stepwise linear regression starting from the model including only the SNP with the lowest individual P value was performed to determine the subset of SNPs with the strongest association. In a final, exploratory stage, we evaluated the association of several biologically relevant gene combinations with ferritin level. To limit the number of combinations tested, we considered only those for which a molecular interaction between gene products has been described and which include one gene showing significant association at the single-gene level. Again, we used multiple-regression models. To evaluate whether more-complex models were significantly better predictors of phenotype than simpler ones, we compared nested models (simpler models are particular cases of the more complex models) using F statistics. These results are exploratory, in the sense that we drop the requirement for multiple-comparison adjustment while assessing significance. For each gene combination, a "best model" was estimated with a backward regression, starting from a full model that included the three SNPs displaying the strongest individual association for each gene and two SNP-interaction terms. SNP rs235756, located in the 3′ region of BMP2, is significantly associated with ferritin level in our C282Y homozygote sample (table 1), after correction for multiple testing (uncorrected P=4.4×10−5; corrected P=.002). Note that the association would still have been significant if a more conservative Bonferroni correction for 75 tests had been applied (corrected P value would then be .003). The T allele associated with higher ferritin level has a frequency of 0.64 (HapMap CEU frequency is 0.58). Mean ferritin level, adjusted for age and sex, is 654.66 ng/ml among TT genotypes, 516.39 ng/ml among TC genotypes, and 349.11 ng/ml among CC genotypes. A neighboring SNP in the gene (rs910141) also displays a suggestive association (corrected P=.062), but rs235756 and rs910141 are in LD (r2=0.528), as can be seen from figure 1. Interestingly, SNP rs235756 has no impact on the age at diagnosis (mean age at diagnosis, adjusted for sex, is 44.7 years among TT genotypes, 45 years among TC genotypes, and 45.8 years among CC genotypes; P value of the analysis of variance [ANOVA] is .82). Suggestive but nonsignificant associations are also observed for one SNP in BMP4 (rs4901474, uncorrected P=.0054, corrected P=.335) and one synonymous SNP in IL6 (rs2069849; uncorrected P=.0051, corrected P=.316). Because the results for the allelic and the genotypic tests were very similar, the following analyses were performed considering the allelic model only.Table 1.Single SNP Genotypic and Allelic Association with Ferritin Level for All 75 SNPsPaP value uncorrected for multiple testing. for Association TestGene (Chromosome) and SNPLocation (bp)MAFGenotypicAllelicAllele (Frequency) Associated with Higher Ferritine LevelHAMP (19): rs91614540,459,724.113.7637.5016 rs1040524640,460,789.221.1045.0423A (.779) rs188269440,463,222.376.3376.1576 rs725143240,467,281.491.8527.5838 rs1297132140,471,262.376.8367.9815 rs1040223340,472,691.347.2110.1180 rs1770518840,473,156.084.9412.8520BMP2 (20): rs22069176,683,511.434.9867.9516 rs2357306,684,189.418.9146.9172 rs60770606,688,317.099.1262.0498T (.099) rs2357106,688,366.439.5832.3072 rs19804996,694,498.495.5092.3742 rs31782506,708,201.173.4460.2532 rs2357726,710,719.409.2757.1124 rs61174326,712,536.233.9039.6946 rs1731076,713,841.380.0026.0052T (.380) rs2357576,714,019.374.1994.0933 rs2357566,715,111.3641.80 × 10−44.42 × 10−5T (.636) rs9101416,715,642.255.0038.0010G (.745) rs2357536,717,533.348.2473.0950 rs60545146,719,370.052.4355.4865 rs2357046,720,263.120.9306.7748 rs178046396,721,316.092.5071.3447BMP4 (14): rs1049846453,441,332.193.3857.2666 rs195186553,442,591.372.5052.5954 rs1115799053,453,695.303.3464.1881 rs374255553,455,219.058.1838.0760 rs214710553,475,815.448.7883.5060 rs444423553,480,669.492.1852.6077 rs76264253,492,803.409.7235.5443 rs195786053,499,105.467.8738.8229 rs657292753,503,140.081.2345.1097 rs1115799453,521,052.076.0766.1883 rs195784453,527,828.230.1068.5648 rs490147453,539,487.429.0050.0054C (.429)HJV (1): rs16827043144,106,797.102.8254.9153 rs7536827144,109,299.452.9444.8876SMAD1 (4): rs6537355146,622,042.149.2329.7810 rs2118438146,647,834.187.1893.0839 rs714195146,665,130.408.9119.7041 rs1016792146,698,229.200.3142.1397 rs2036138146,704,312.467.7503.8810 rs11939979146,707,777.469.6741.7925SMAD4 (18): rs60607346,749,479.440.3876.7981 rs1016378946,749,360.070.7042.7042 rs1050291346,822,269.269.9241.9260 rs1766388746,843,716.098.5783.3150 rs1766399446,920,796.329.3553.8460 rs930440846,928,354.362.0510.3874 rs996387846,933,520.089.1652.0655 rs724245946,935,348.396.6693.3740SMAD5 (5): rs2346361135,476,404.459.2769.2209 rs9327744135,501,661.236.4137.2589TFR2 (7): rs7812235100,049,422.195.5966.3716 rs10247962100,057,865.155.2861.7993 rs4434553100,078,127.492.0920.0315G (.492)SLC40A1 (2): rs12693541190,126,935.134.1990.0757 rs11884632190,133,087.244.6722.4917 rs2304704190,138,422.354.1747.1206 rs10188230190,140,858.020.3613.3698 rs16831659190,141,534.103.2471.1823 rs10202029190,154,529.025.0677.0677 rs2352267190,157,210.395.7741.4807 rs1123109190,162,978.208.8950.6928IL6 (7): rs188024222,726,132.494.4962.8671 rs1049956322,727,013.238.2078.0782 rs205657622,727,727.307.2417.0924 rs1270038622,729,534.177.4979.5244 rs206982722,731,981.106.8888.6857 rs180079522,733,170.441.9075.7436 rs206983722,734,552.068.2151.0796 rs206984022,735,097.344.8775.6220 rs206986022,737,563.088.2467.1021 rs206984922,737,681.007.0051.0051T (.007) rs206986122,738,179.018.3537.3537a P value uncorrected for multiple testing. Open table in a new tab Results for haplotype association are presented in table 2. In BMP2, the best haplotype combination provided a P value of only .0098, which did not improve the single-SNP association. Similar results were observed for BMP4 and IL6. Tests based on unphased genotype data did not detect any additional interesting SNP combination (results not shown). However, the tagging strategy chosen leads to a low LD among SNPs in each gene (mean r2=0.1), a situation in which multimarker approaches (either phased or unphased) are expected to be less powerful.32Clayton D Chapman J Cooper J Use of unphased multilocus genotype data in indirect association studies.Genet Epidemiol. 2004; 27: 415-428Crossref PubMed Scopus (165) Google ScholarTable 2.Haplotype Association with Ferritin LevelGeneNo. of SNPs in the GeneGlobal PaGlobal P values obtained by permutation are not corrected for multiple testing across genes but are corrected for the number of SNPs included in each haplotype.Genes with 10 SNPsbGlobal P value for the best five-SNP combinatio
Referência(s)