Genetic Variants in Major Histocompatibility Complex-Linked Genes Associate With Pediatric Liver Transplant Rejection
2008; Elsevier BV; Volume: 135; Issue: 3 Linguagem: Inglês
10.1053/j.gastro.2008.05.080
ISSN1528-0012
AutoresRakesh Sindhi, Brandon W. Higgs, Daniel E. Weeks, Chethan Ashokkumar, Ronald Jaffe, Cecilia Kim, Patrick Wilson, Nydia Chien, Joseph Glessner, Anjan Kumar Talukdar, George Mazariegos, M. Michael Barmada, Edward Frackleton, Nancy Petro, Andrew W. Eckert, Hákon Hákonarson, Robert E. Ferrell,
Tópico(s)Liver Disease Diagnosis and Treatment
ResumoBackground & Aims: Limited access to large samples precludes genome-wide association studies of rare but complex traits. To localize candidate genes with family-based genome-wide association, a novel exploratory analysis was first tested on 1774 major histocompatibility complex single nucleotide polymorphisms (SNPs) in 240 DNA samples from 80 children with primary liver transplantation and their biologic parents. Methods: Initially, 57 SNPs with large differences (P < .05) in minor allele frequencies were selected when parents of children with early rejection (rejectors) were compared with parents of nonrejectors. Results: In hypothesis testing of selected SNPs, the gamete competition statistic identified the minor allele G of the SNP rs9296068, near HLA-DOA, as being significantly different (P = .018) between outcome groups in parent-to-child transmission. Subsequent simple association testing confirmed over- and undertransmission of rs9296068 based on the most significant differences between outcome groups, of 1774 SNPs tested (P = .002), and allele (G) frequencies that were greater among rejectors (51.4% vs 36.8%, respectively, P = .015) and lower among nonrejectors (26.8% vs 36.8%, respectively, P = .074) compared with 400 normal control Caucasian children. In early functional validation, rejectors demonstrated significant repression of the first HLA-DOA exon closest to rs9296068. Also, intragraft B lymphocytes, whose antigen-presenting function is selectively inhibited by HLA-DOA were 3-fold more numerous during rejection among rejectors with the risk allele, than those without. Conclusions: The minor allele of the SNP rs9296068 is significantly associated with liver transplantation rejection and with enhanced B-lymphocyte participation in rejection, likely because of a dysfunctional HLA-DOA gene product. Background & Aims: Limited access to large samples precludes genome-wide association studies of rare but complex traits. To localize candidate genes with family-based genome-wide association, a novel exploratory analysis was first tested on 1774 major histocompatibility complex single nucleotide polymorphisms (SNPs) in 240 DNA samples from 80 children with primary liver transplantation and their biologic parents. Methods: Initially, 57 SNPs with large differences (P < .05) in minor allele frequencies were selected when parents of children with early rejection (rejectors) were compared with parents of nonrejectors. Results: In hypothesis testing of selected SNPs, the gamete competition statistic identified the minor allele G of the SNP rs9296068, near HLA-DOA, as being significantly different (P = .018) between outcome groups in parent-to-child transmission. Subsequent simple association testing confirmed over- and undertransmission of rs9296068 based on the most significant differences between outcome groups, of 1774 SNPs tested (P = .002), and allele (G) frequencies that were greater among rejectors (51.4% vs 36.8%, respectively, P = .015) and lower among nonrejectors (26.8% vs 36.8%, respectively, P = .074) compared with 400 normal control Caucasian children. In early functional validation, rejectors demonstrated significant repression of the first HLA-DOA exon closest to rs9296068. Also, intragraft B lymphocytes, whose antigen-presenting function is selectively inhibited by HLA-DOA were 3-fold more numerous during rejection among rejectors with the risk allele, than those without. Conclusions: The minor allele of the SNP rs9296068 is significantly associated with liver transplantation rejection and with enhanced B-lymphocyte participation in rejection, likely because of a dysfunctional HLA-DOA gene product. In children, liver transplantation (LTx) is usually performed for multiple congenital liver diseases and results in highly variable outcomes.1Martin S.R. Atkison P. Anand R. et al.SPLIT Research GroupStudies of pediatric liver transplantation 2002: patient and graft survival and rejection in pediatric recipients of a first liver transplant in the United States and Canada.Pediatr Transplant. 2004; 8: 273-283Crossref PubMed Scopus (130) Google Scholar Acute cellular rejection occurs in 50% and post-LTx lymphoma-like malignancy affects 2%–10% of patients.2Jain A. Mazariegos G. Kashyap R. et al.Pediatric liver transplantation A single center experience spanning 20 years.Transplantation. 2002; 73: 941-947Crossref PubMed Scopus (112) Google Scholar Genetic variants, exemplified most commonly by single nucleotide polymorphisms (SNPs), are a significant basis for individual variation.3Ireland J. Carlton V.E.H. Falkowski M. et al.Large-scale characterization of public database SNPs causing non-synonymous changes in three ethnic groups.Hum Genet. 2006; 119: 75-83Crossref PubMed Scopus (14) Google Scholar However, the majority of the >10 million catalogued SNPs do not alter gene function.4Sherry S.T. Ward M.H. Kholodov M. et al.dbSNP: the NCBI database of genetic variation.Nucleic Acids Res. 2001; 29 (Available at: www.ncbi.nlm.nih.gov/pubmed/11125122. Accessed July, 2008): 308-311Crossref PubMed Scopus (4643) Google Scholar In addition, promising associations involving discrete SNPs are rarely reproduced.5Hutchinson I.V. Pravica V. Sinnot P.J. Genetic regulation of cytokine synthesis: consequences on acute and chronic organ allograft rejection.Graft. 1998; 1: 15Google Scholar, 6Daly A.K. Day C.P. Donaldson P.T. 2002 Polymorphisms in immunoregulatory genes: toward individualized immunosuppressive therapy?.Am J Pharmacogenomics. 2002; 2: 13-23Crossref PubMed Scopus (22) Google Scholar, 7Hirschhorn J.N. Lohmueller K. Byrne E. et al.A comprehensive review of genetic association studies.Genet Med. 2002; 4: 45-61Crossref PubMed Scopus (1387) Google ScholarPopulation stratification, or overrepresentation of one ethnicity within an outcome group, may allow an SNP representing this ethnicity to be viewed as the outcome-specific SNP.8Cardon L.R. Palmer L.J. Population stratification and spurious allelic association.Lancet. 2003; 361: 598-604Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar Family-based association studies can minimize stratification.9Spielman R.S. McGinnis R.E. Ewens W.J. Transmission test for linkage disequilibrium: the insulin gene region and insulin-dependent diabetes mellitus (IDDM).Am J Hum Genet. 1993; 52: 506-516PubMed Google Scholar In this design, parental genotype serves as the control, and transmission frequency of an SNP from heterozygous parents to affected offspring is compared with the expected 50% transmission frequency. Significant deviations reflect transmission disequilibrium and are more likely to represent disease-specific variation than variations because of ethnicity. The known location of such an SNP is then used to identify a potential candidate gene and a causal variant in proximity. Increasingly, genome-wide association studies are being conducted, recognizing that most disease traits are complex and likely originate from an interaction between multiple causal variants within multiple genes and the environment. Several limitations preclude application of genome-wide association in transplantation, especially among pediatric LTx recipients. First, simultaneous testing of large numbers of SNPs necessitates stringent multiple-testing correction. Under these conditions, statistical power requirements mandate a large subject sample or a 2-step study in which candidate SNPs identified in a screening sample are replicated in a second step. Accruing a homogeneous validation cohort can take several years because approximately 550 pediatric LTx are performed annually in the United States and are distributed among >50 transplant centers.102007 OPTN/SRTR Annual Report 1997-2006. HHS/HRSA/HSB/DOT.Google Scholar Second, the numbers of candidate SNPs in an association study may not exceed false-discovery thresholds. From such a limited list of candidates, any true-positive association may at best account for large effects. We report for the first time a novel, multistep approach to candidate gene localization, which incorporates preliminary functional validation in the same test cohort. First, a 2-tier, family-based association method identifies the candidate gene or locus. Next, transmission distortion in that locus is confirmed by allele frequency comparisons with a large control group. Because SNPs within noncoding regions can influence gene expression and splicing at distances up to 100 kilobase (kb), differential regulation and alternative splice variants of candidate genes are evaluated with probes specific to exons of whole gene transcripts in the first of 2 preliminary functional validation tests.11Hull J. Campino S. Rowlands K. et al.Identification of common genetic variation that modulates alternative splicing.PLoS Genet. 2007; 3: e99Crossref PubMed Scopus (124) Google Scholar, 12Chen Y. Zhu J. Lum P.Y. et al.Variations in DNA elucidate molecular networks that cause disease.Nature. 2008; 452: 429-435Crossref PubMed Scopus (724) Google Scholar In the second test, the candidate is characterized in diseased tissue with immunohistochemistry. Because our sample size is small and consists of 80 case-parent-parent trios, our association method is based on the following biologic assumptions: (1) If an SNP is strongly associated with an inherited trait, it should demonstrate differences in parental allele frequencies (PAF) between the 2 groups; (2) In family-based association testing, a genetic variant that is associated with rejection/nonrejection should show overtransmission in one outcome group and undertransmission in the other in the gamete competition test (GC). The GC statistic evaluates transmission disequilibrium using full pedigree data, similar to the well-known Transmission Disequilibrium Test,8Cardon L.R. Palmer L.J. Population stratification and spurious allelic association.Lancet. 2003; 361: 598-604Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar but is based on a likelihood ratio test.13Sinsheimer J.S. Blangero J. Lange K. 2000. Gamete-competition models.Am J Hum Genet. 2000; 66: 1168-1172Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar The GC statistic also handles missing data and allows haplotype-based analysis. (3) In a confirmatory step, potential candidates should also demonstrate (a) significant differences in allele frequency when the 2 outcome groups are compared with each other and (b) evidence of overtransmission in one group and undertransmission in the other when compared individually, with a large cohort of 400 normal Caucasian children. We asked whether rejection/nonrejection outcomes are associated with genetic variants in the major histocompatibility complex (MHC) region. The 1774 MHC-SNPs evaluated here are a subset of 550,000 genome-wide SNPs recently characterized in our test population. MHC-SNPs have been analyzed separately at first to explore the utility of our statistical method, which is based in part on screening/testing approaches for quantitative traits proposed by others, by applying it to a candidate region with known impact in organ transplantation.14Ionita-Laza I. McQueen M.B. Laird N.M. et al.Genome-wide weighted hypothesis testing in family-based association studies, with an application to a 100K scan.Am J Hum Genet. 2007; 81: 607-614Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 15Shih M.C. Whittemore A.S. Tests for genetic association using family data.Genet Epidemiol. 2002; 22: 128-145Crossref PubMed Scopus (30) Google Scholar, 16Van Steen K. McQueen M.B. Herbert A. et al.Genomic screening and replication using the same data set in family-based association testing.Nat Genet. 2005; 37: 683-691Crossref PubMed Scopus (148) Google Scholar, 17Sheldon S. Poulton K. HLA typing and its influence on organ transplantation.Methods Mol Biol. 2006; 333: 157-174PubMed Google Scholar The results show that the minor allele G at the SNP locus rs9296068, which is significantly associated with rejection outcomes, localizes to the 5′ untranslated region (UTR) region of the HLA-DOA gene. Differential regulation of HLA-DOA, which is selectively expressed in B lymphocytes, is suggested by associated repression of the first HLA-DOA exon and significantly higher B-lymphocyte content in allograft biopsy specimens from rejectors.18Sindhi R. Magill A. Abdullah A. et al.Enhanced donor-specific alloreactivity occurs independent of immunosuppression in children with early liver allograft rejection.Am J Transplant. 2005; 5: 96-102Crossref PubMed Scopus (20) Google Scholar, 19Denzin L.K. Sant'Angelo D.B. Hammond C. et al.Negative regulation by HLA-DO of MHC class II-restricted antigen processing.Science. 1997; 278: 106-109Crossref PubMed Google Scholar All studies were performed with approval from the University of Pittsburgh Institutional Review Board. DNA was extracted from 3-mL whole blood samples from 80 primary pediatric LTx recipients ages 0–22 years and their biologic parents (240 DNA samples). All children received previously described tacrolimus monotherapy after steroid-free induction with rabbit, anti-human thymocyte globulin (rATG; Genzyme, Cambridge, MA).20Roucard C. Thomas C. Pasquier M.A. et al.In vivo and in vitro modulation of HLA-DM and HLA-DO is induced by B lymphocyte activation.J Immunol. 2001; 167: 6849-6858Crossref PubMed Scopus (32) Google Scholar Children who experienced biopsy-proven acute cellular rejection within the first 60 days after LTx were termed rejectors. Normal controls consisted of 400 disease-free Caucasian children with no prior or ongoing use of immunosuppressants or transplantation. In addition, all 400 samples were selected for the absence of large copy number variants that could suggest a disease association. This normal control population was recruited at the Center for Applied Genomics at the Children's Hospital of Philadelphia. Genotyping was performed with 550,000 genome-wide SNP loci with the HumHap550k SNP bead array (Illumina, San Diego, CA). Analysis from this experiment was restricted to 1813 SNPs covering 168 MHC genes on chromosome 6p, of which 1774 passed our quality control criteria. Genotype and gene expression data can be found at http://www.ncbi.nlm.nih.gov/projects/geo/query/acc.cgi?acc=GSE11361; accession No.: GSE11361 . DNA extracted by the Gentra Purigene system (Minneapolis, MN) was activated, and SNPs were characterized using manufacturer's protocol. The Illumina Beadstation GX software was used to extract genotype calls (Illumina).21Shen R. Fan J.B. Campbell D. et al.High-throughput SNP genotyping on universal bead arrays.Mutat Res. 2002; 573: 70-82Crossref Scopus (319) Google Scholar A total of 1813 SNPs from the MHC region of the HumHap550k SNP array (Illumina) were identified for analysis. Genotype data from the array were merged with pedigree and phenotype data and quality controlled for the following: Mendelian errors (recoded to missing), Hardy–Weinberg equilibrium (SNPs with deviations at P < .001 removed), missing rate across patients (>10% removed), minimal MAF (SNPs with MAF <1% removed), and low genotyping rate for an individual (<10% removed in the simple association test) using the PLINK v0.99r software package.22Purcell S, Neale B, Todd-Brown K, et al. PLINK: a toolset for whole-genome association and population-based linkage analysis. Am J Hum Genet. Available at: http://pngu.mgh.harvard.edu/purcell/plink/. Accessed July 2008.Google Scholar This filtering reduced the number of SNPs to 1774 total. A 2-sample proportions statistic (2-sided) was used to determine PAF differences (P value cut-off < .05) between parents of rejectors and nonrejectors. This screening step yielded a subset of MHC-SNPs for subsequent hypothesis testing using the GC statistic, as implemented in the software package Mendel v7.0.0.23Lange K. Cantor R. Horvath S. et al.Mendel version 4.0: a complete package for the exact genetic analysis of discrete traits in pedigree and population data sets.Am J Hum Genet. 2001; 69: A1886Google Scholar In this approach, a segregation parameter (τ) is estimated by maximum likelihood from the family data, and a likelihood ratio test is then applied. For a 2-allele locus with alleles 1 and 2, the probability that a heterozygous parent with genotype 1/2 transmits allele 1 is defined as Pr (1/2 -> 1) = τ1/(τ1+ τ2); the null hypothesis of Mendelian transmission corresponds to τ1 = τ2 = 1.14Ionita-Laza I. McQueen M.B. Laird N.M. et al.Genome-wide weighted hypothesis testing in family-based association studies, with an application to a 100K scan.Am J Hum Genet. 2007; 81: 607-614Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar The transmission parameter τk is set to 1 for the most frequent allele k, whereas the remaining τk values (for all k not equal to the most frequent allele) are estimated by maximum likelihood. In our application, we assumed symmetric transmission: that is, an allele that is overtransmitted to rejectors is assumed to be similarly undertransmitted to nonrejectors. Results are summarized as the P value and χ2 statistic (with 95% confidence bounds) for the PAF comparison and the estimated segregation parameter (τ), the allele frequency, and the P value of the GC statistic (Appendix 1). Note that this screening/testing approach markedly reduces multiple testing problems because the PAF test (which only uses parental genotypes) is independent of the GC test (which depends on how often specific alleles are transmitted to the children). Thus, because the PAF test alone is used for SNP selection, we only have to adjust for the actual number of GC tests conducted on the much smaller number of selected SNPs. Comparisons of minor allele frequency (MAF) for significant MHC-SNPs were conducted between rejectors and normal controls and between nonrejectors and normal controls (χ2 test). The Affymetrix (Santa Clara, CA) Human Exon 1.0 ST array was used to measure differential splicing patterns in archived RNA isolated from 29 of 80 children. Probe summaries for both the genes and the exons were computed using the Affymetrix Power Tools software and “rma-sketch” normalization method. The gene-level normalized intensities were computed with the MiDAS algorithm, and both the splicing index24Srinivasan K. Shiue L. Hayes J.D. et al.Detection and measurement of alternative splicing using splicing-sensitive microarrays.Methods. 2005; 37: 345-359Crossref PubMed Scopus (87) Google Scholar and the Student t test P values (2-sided) were computed on the normalized intensities values in R.25R Development Core Team. R: a language and environment for statistical computing. Vienna: R Foundation for Statistical Computing, Vienna, Austria. Available at: http://www.R-project.org. Accessed July 2008.Google Scholar Principal components analysis was calculated separately on exon-level and gene-level intensities and was used to remove 3 outlier samples, for a remaining total of 26 samples: 11 rejectors and 15 nonrejectors. To remove low expressed gene-level probes, those with values less than 3.5 (log2 scale) in >50% of the samples in either group were filtered out, leaving 17,242 of the original 22,011 probes. Those gene-level probes that were highly differentially expressed between groups were also removed using a fold change threshold of log2 (10). This step accounts for the tendency for the gene-level probe set intensities in each group to be “disproportionately affected by background noise or saturation” (Affymetrix Technical notes). Exon-level probes were filtered based on Affymetrix Power Tools detection above background P value greater than .05 in n-1 samples leaving 218,402 of the original 287,329 exon-level probes. After gene and exon probe filtering, 7 gene-level probes (and 67 corresponding exon-level probes for the genes) within a window size of 200 kb that included the HLA-DOA gene on chromosome 6 were extracted and examined for differential splicing (positions 33,000,000–33,200,000). This was conducted to restrict the analysis to only the immediate region surrounding the HLA-DOA gene. Because the HLA-DOA gene inhibits class II antigen presentation selectively in B cells, we asked whether B cells were present in allograft biopsies showing rejection, and whether the B-cell content of allograft biopsy tissue varied with the presence or absence of the risk allele of rs9296068. Slides were recut from stored tissue blocks for 36 of 80 children, with 4-μm-thick tissue sections, stained in batches on a Ventana immunostainer using mouse monoclonal anti-CD79a (DAKO M7050 at 1:100, Ventana CCI mild, 32 minutes at 32°C, iView DAB detection), as well as their positive and negative biologic staining controls for each batch. Hematoxylin counterstain was applied after DAB detection. The surface marker CD79a identifies cells of B-cell/plamacytoid lineage. Cell counting was done blinded to outcome and prior to histologic re-review of the tissues. Duplicate counts were done on each tenth case to assess variance. Portal and lobular regions of allografts were optically scored separately, at magnifications of ×400 and ×200, respectively. B-cell counts were expressed as total number per portal area and total number per lobule (Figure 3). Early acute cellular rejection occurred at mean ± SD 32.5 ± 4.2 days after LTx in 37 children who were termed rejectors. Rejectors and nonrejectors (n = 43) were similar with respect to age (median ± SEM, 6 ± 1.1 vs 4.6 ± 0.9 years, respectively, P = NS), male:female sex distribution (16:21 vs 21:22, respectively, P = NS), and etiology of liver failure requiring LTx (Table 1). The racial distribution in the rejector and nonrejector groups was (Caucasian:African American:other, 31:2:4 vs 42:0:1, respectively). Also, there were no differences between rejectors and nonrejectors in donor-recipient matching at the HLA-A, HLA-B, and HLA-DR loci (1.4 ± 1.2 vs 1.8 ± 1.2 antigens, respectively, P = NS) or disease severity as reflected in the Pediatric End-Stage Liver Disease score (25.5 ± 13.2 vs 24 ± 14.2, respectively, P = NS). The sample size was too small to evaluate the effect of disease, age at transplantation, and immunosuppression on rejection outcomes or whether the highly associated SNP(s) were independent predictors of rejection outcome.Table 1Etiology of Liver Disease Leading to LTx in 80 ChildrenEtiology of liver diseaseRejectorsNonrejectorsAllagilles11Autoimmune hepatitis30Biliary atresia813BRIC: Benign recurrent intrahepatic cholestasis01Budd–Chiari syndrome01Bylers disease PFIC type I10Carolis disease10Congenital hepatic fibrosis12Crigler–Najjar syndrome15Cystic fibrosis11Fulminant hepatic failure22Giant cell hepatitis01Glycogen storage disease10Hepatic fibrosis10Hepatoblastoma11Hepatocellular carcinoma01Methylmalonic acidemia10Maple syrup urine disease78Ornithin transcarbamylase deficiency12Primary sclerosing cholangitis21Secondary biliary cirrhosis01Paucity of bile ducts10Tyrosenemia11Cirrhosis, unknown21Total3743 Open table in a new tab Fifty-seven SNPs passed our screening test because of large differences in MAF when parents of rejectors were compared with parents of nonrejectors (Appendix 1). When the GC statistic was applied to these 57 SNPs, only 1 SNP, rs9296068, in the 5′ flanking UTR of HLA-DOA demonstrated differences between groups with P < .05 in parent-to-child transmission (P = .0183, Table 2). Specifically, the minor allele G was transmitted more frequently to rejectors and less so to nonrejectors from biologic parents. The physical position of the implicated SNP is 33096673 (Build 35). Figure 1 summarizes the results of the PAF comparison for the entire set of 1774 MHC SNPs (Figure 1, upper panel), and the results of the GC test applied to the 57 selected SNPs for rejectors and nonrejectors (Figure 1, lower panel).Table 2Parental Allele Frequencies and GC Transmission Characteristics of HLA-DOA SNP rs9296068Rejector vs nonrejector MAF P value.041Rejector vs nonrejector GC P value.018GC transmission parameter (τ2)aFor the GC statistic, under the null hypothesis of Mendelian segregation, τk = 1 for all k alleles.111.9Allele2Rejector parents MAF46.3% Number transmitted19 Number nontransmitted12Nonrejector parents MAF33.8% Number transmitted7 Number nontransmitted17NOTE. Only 1 SNP, rs9296068 near the HLA-DOA gene, met criteria for selection and hypothesis testing in our 2-tier approach. This SNP demonstrated a large difference (P = .041) in MAF when parents of rejectors were compared with parents of nonrejectors, as well as significant differences in parent-to-child transmission between rejectors and nonrejectors, on the GC test (P = .0183). Transmission data show more parents (n = 19) transmitting the minor allele of rs9296068 to their rejector offspring than parents who did not (n = 12). The reverse was true among nonrejectors, whose parents were less likely to transmit this risk allele (7 transmissions vs 17 nontransmissions).MAF, minor allele frequency.a For the GC statistic, under the null hypothesis of Mendelian segregation, τk = 1 for all k alleles.11Hull J. Campino S. Rowlands K. et al.Identification of common genetic variation that modulates alternative splicing.PLoS Genet. 2007; 3: e99Crossref PubMed Scopus (124) Google Scholar Open table in a new tab NOTE. Only 1 SNP, rs9296068 near the HLA-DOA gene, met criteria for selection and hypothesis testing in our 2-tier approach. This SNP demonstrated a large difference (P = .041) in MAF when parents of rejectors were compared with parents of nonrejectors, as well as significant differences in parent-to-child transmission between rejectors and nonrejectors, on the GC test (P = .0183). Transmission data show more parents (n = 19) transmitting the minor allele of rs9296068 to their rejector offspring than parents who did not (n = 12). The reverse was true among nonrejectors, whose parents were less likely to transmit this risk allele (7 transmissions vs 17 nontransmissions). MAF, minor allele frequency. In the confirmatory association testing step, 77 SNPs showed significant differences in allele frequencies when rejectors were compared directly with nonrejectors (P ≤ .05). Among these 77 SNPs, additional between-group comparisons showed 39 SNPs to be significantly different among rejectors and 19 SNPs to be significantly different among nonrejectors when each group was compared separately with 400 normal controls (Appendix 2). In direct comparisons, the differences between rejectors and nonrejectors were most significant (P = .002) for the SNP rs9296068 (Table 3). When compared with normal controls, the minor allele (G) of rs9296068 was more commonly seen among rejectors (36.7% vs 51.4%, respectively, P = .015) but less commonly among nonrejectors (36.7 % vs 26.8%, respectively, P = .074). Only 1 other SNP, rs9276994, shows similar differences in distribution when rejectors are compared with nonrejectors (48.6% vs 28%, respectively, P = .009) and when normal controls are compared either with rejectors (37.7% vs 48.6%, respectively, P = .074) or with nonrejectors (37.7% vs 28%, respectively, P = .083) (Table 4). In all, 5 of 14 top-ranked discriminatory SNPs localized to the 5′ flanking UTR of HLA-DOA (Table 4).Table 3Results of Confirmatory Association Testing of rs9296068Rejector vs controls MAF in rejector children51.4% MAF in controls36.8% χ25.90 P value.015 Odds ratio1.82Nonrejector vs controls MAF in nonrejector children26.8% MAF in controls36.8% χ23.18 P value.074 Odds ratio0.63NOTE. Confirmatory association testing shows that MAF for rs9296068 are greater among rejectors, and less among nonrejectors, compared with a large cohort of 400 normal Caucasian children. Together, these results confirm parent-to-child overtransmission among rejectors and undertransmission among nonrejectors observed in the GC test. MAF differences are based on the samples used. For the unrelated case/control association shown in the Table above, children are used to calculate allele frequencies. For the GC test in Table 2, trio information is used, resulting in minor differences in allele frequency.MAF, minor allele frequency. Open table in a new tab Table 4Results of Confirmatory Association Testing for SNPsSNPPhysical locationClosest geneAllele frequencyχ2 test, P valuesR, %NR, %NC, %R vs NCNR vs NCR vs NRrs251790429,976,963HCG2P72.915.97.5.147.009.007rs313487930,123,884ZNRD126.648.741.1.023.192.007rs936675230,132,656ZNRD134.315.920.0.005.368.008rs926144130,199,737TRIM3115.73.713.6.623.01.01rs134522930,290,374TRIM2614.32.411.9.553.009.007rs126458330,401,462TRIM3914.32.45.0.001.3.007rs126458130,405,484TRIM3916.23.75.3.000.524.009rs309409730,741,854DHX1614.32.49.8.228.028.007rs60287532,681,607HLA-DRB142.922.023.8.000.707.006rs645769933,089,625HLA-DOA54.331.746.8.226.009.005rs927699433,092,233HLA-DOA48.628.137.8.075.083.009rs693399433,095,098HLA-DOA57.136.346.0.614.002.01rs929606833,096,673HLA-DOA51.426.836.8.015.074.002rs927701533,101,244HLA-DOA44.324.437.0.228.023.01NOTE. Results of confirmatory association testing for SNPs, ordered by physical position, which showed the most significant differences in MAF (P ≤ .010) between rejectors and nonrejectors (last column). Of 14 such SNPs, 5 are near HLA-DOA. Among those, rs9296068 and rs9276994 show MAF, which are greater among rejectors, and less among nonrejectors, compared with 400 normal control Caucasian children (NC). Only rs9296068 shows differences in PAF between outcome groups, satisfying selection criteria for candidacy, whereas rs9276994 fails the PAF test.R, rejectors; NR, nonrejectors; NC, normal control Caucasian children. Open table in a new tab NOTE. Confirmato
Referência(s)