Identifying rare variants for genetic risk through a combined pedigree and phenotype approach: application to suicide and asthma.

Suicidal behavior is a complex disorder, with evidence for genetic risk independent of other genetic risk factors including psychiatric disorders. Since 1996, over 3000 DNA samples from Utah suicide decedents have been collected and banked for research use through the Utah Medical Examiner. In addition, over 12,000 Utah suicides were identified through examination of death certificates back to 1904. By linking this data with the Utah Population Database, we have identified multiple extended pedigrees with increased risk for suicide completion. A number of medical conditions co-occur with suicide, including asthma, and this study was undertaken to identify genetic risk common to asthma and suicide. This study tests the hypothesis that a particular comorbid condition may identify a more homogeneous genetic subgroup, facilitating the identification of specific genetic risk factors in that group. From pedigrees at increased risk for suicide, we identified three pedigrees also at significantly increased familial risk for asthma. Five suicide decedents from each of these pedigrees, plus an additional three decedents not from these pedigrees with diagnosed asthma, and 10 decedents with close relatives with asthma were genotyped. Results were compared with 183 publicly available unaffected control exomes from 1000 Genomes and CEPH (Centre d'etude du polymorphisme humain) samples genotyped on the same platform. A further 432 suicide decedents were also genotyped as non-asthma suicide controls. Genotyping was done using the Infinium HumanExome BeadChip. For analysis, we used the pedigree extension of Variant Annotation, Analysis and Search Tool (pVAAST) to calculate the disease burden of each gene. The Phenotype Driven Variant Ontological Re-ranking tool (Phevor) then re-ranked our pVAAST results in context of the phenotype. Using asthma as a seed phenotype, Phevor traversed biomedical ontologies and identified genes with similar biological properties to those known to result in asthma. Our top associated genes included those related to neurodevelopment or neural signaling (brain-derived neurotrophic factor (BDNF), neutral sphingomyelinase 2 (SMPD2), homeobox b2 (HOXB2), neural cell adhesion molecule (NCAM2), heterogeneous nuclear ribonucleoprotein A0 (HNRNPA0)), inflammation (free fatty acid receptor 2 (FFAR2)) and inflammation with additional evidence of neuronal involvement (oxidized low density lipoprotein receptor 1 (OLR1), toll-like receptor 3 (TLR3)). Of particular interest, BDNF has been previously implicated in both psychiatric disorders and asthma. Our results demonstrate the utility of combining pedigree and co-occurring phenotypes to identify rare variants associated with suicide risk in conjunction with specific co-occurring conditions.

Genetic risk factors in two Utah pedigrees at high risk for suicide.

We have used unique population-based data resources to identify 22 high-risk extended pedigrees that show clustering of suicide over twice that expected from demographically adjusted incidence rates. In this initial study of genetic risk factors, we focused on two high-risk pedigrees. In the first of these (pedigree 12), 10/19 (53%) of the related suicides were female, and the average age at death was 30.95. In the second (pedigree 5), 7/51 (14%) of the suicides were female and the average age at death was 36.90. Six decedents in pedigree 12 and nine in pedigree 5 were genotyped with the Illumina HumanExome BeadChip. Genotypes were analyzed using the Variant Annotation, Analysis, and Search program package that computes likelihoods of risk variants using the functional impact of the DNA variation, aggregative scoring of multiple variants across each gene and pedigree structure. We prioritized variants that were: (1) shared across pedigree members, (2) rare in other Utah suicides not related to these pedigrees, (3) < or = 5% in genotyping data from 398 other Utah population controls and (4) < or = 5% frequency in publicly available sequence data from 1358 controls and/or in dbSNP. Results included several membrane protein genes (ANO5, and TMEM141 for pedigree 12 and FAM38A and HRCT1 for pedigree 5). Other genes with known neuronal involvement and/or previous associations with psychiatric conditions were also identified, including NFKB1, CASP9, PLXNB1 and PDE11A in pedigree 12, and THOC1, and AUTS2 in pedigree 5. Although the study is limited to variants included on the HumanExome BeadChip, these findings warrant further exploration, and demonstrate the utility of this high-risk pedigree resource to identify potential genes or gene pathways for future development of targeted interventions.

Immunophenotypic and gene expression analysis of monoclonal B-cell lymphocytosis shows biologic characteristics associated with good prognosis CLL.

Monoclonal B-cell lymphocytosis (MBL) is a hematologic condition wherein small B-cell clones can be detected in the blood of asymptomatic individuals. Most MBL have an immunophenotype similar to chronic lymphocytic leukemia (CLL), and 'CLL-like' MBL is a precursor to CLL. We used flow cytometry to identify MBL from unaffected members of CLL kindreds. We identified 101 MBL cases from 622 study subjects; of these, 82 individuals with MBL were further characterized. In all, 91 unique MBL clones were detected: 73 CLL-like MBL (CD5(+)CD20(dim)sIg(dim)), 11 atypical MBL (CD5(+)CD20(+)sIg(+)) and 7 CD5(neg) MBL (CD5(neg)CD20(+)sIg(neg)). Extended immunophenotypic characterization of these MBL subtypes was performed, and significant differences in cell surface expression of CD23, CD49d, CD79b and FMC-7 were observed among the groups. Markers of risk in CLL such as CD38, ZAP70 and CD49d were infrequently expressed in CLL-like MBL, but were expressed in the majority of atypical MBL. Interphase cytogenetics was performed in 35 MBL cases, and del 13q14 was most common (22/30 CLL-like MBL cases). Gene expression analysis using oligonucleotide arrays was performed on seven CLL-like MBL, and showed activation of B-cell receptor associated pathways. Our findings underscore the diversity of MBL subtypes and further clarify the relationship between MBL and other lymphoproliferative disorders.

The sumLINK statistic for genetic linkage analysis in the presence of heterogeneity.

We present the "sumLINK" statistic--the sum of multipoint LOD scores for the subset of pedigrees with nominally significant linkage evidence at a given locus--as an alternative to common methods to identify susceptibility loci in the presence of heterogeneity. We also suggest the "sumLOD" statistic (the sum of positive multipoint LOD scores) as a companion to the sumLINK. sumLINK analysis identifies genetic regions of extreme consistency across pedigrees without regard to negative evidence from unlinked or uninformative pedigrees. Significance is determined by an innovative permutation procedure based on genome shuffling that randomizes linkage information across pedigrees. This procedure for generating the empirical null distribution may be useful for other linkage-based statistics as well. Using 500 genome-wide analyses of simulated null data, we show that the genome shuffling procedure results in the correct type 1 error rates for both the sumLINK and sumLOD. The power of the statistics was tested using 100 sets of simulated genome-wide data from the alternative hypothesis from GAW13. Finally, we illustrate the statistics in an analysis of 190 aggressive prostate cancer pedigrees from the International Consortium for Prostate Cancer Genetics, where we identified a new susceptibility locus. We propose that the sumLINK and sumLOD are ideal for collaborative projects and meta-analyses, as they do not require any sharing of identifiable data between contributing institutions. Further, loci identified with the sumLINK have good potential for gene localization via statistical recombinant mapping, as, by definition, several linked pedigrees contribute to each peak.

Identifying rarer genetic variants for common complex diseases: diseased versus neutral discovery panels.

The power of genetic association studies to identify disease susceptibility alleles fundamentally relies on the variants studied. The standard approach is to determine a set of tagging-SNPs (tSNPs) that capture the majority of genomic variation in regions of interest by exploiting local correlation structures. Typically, tSNPs are selected from neutral discovery panels - collections of individuals comprehensively genotyped across a region. We investigated the implications of discovery panel design on tSNP performance in association studies using realistically-simulated sequence data. We found that discovery panels of 24 sequenced 'neutral' individuals (similar to NIEHS or HapMap ENCODE data) were sufficient to select well-powered tSNPs to identify common susceptibility alleles. For less common alleles (0.01-0.05 frequency) we found neutral panels of this size inadequate, particularly if low-frequency variants were removed prior to tSNP selection; superior tSNPs were found using panels of diseased individuals. Only large neutral panels (200 individuals) matched diseased panel performance in selecting well-powered tSNPs to detect both common and rarer alleles. The 1000 Genomes Project initiative may provide larger neutral panels necessary to identify rarer susceptibility alleles in association studies. In the interim, our results suggest investigators can boost power to detect such alleles by sequencing diseased individuals for tSNP selection.

Shared genomic segment analysis. Mapping disease predisposition genes in extended pedigrees using SNP genotype assays.

We examine the utility of high density genotype assays for predisposition gene localization using extended pedigrees. Results for the distribution of the number and length of genomic segments shared identical by descent among relatives previously derived in the context of genomic mismatch scanning are reviewed in the context of dense single nucleotide polymorphism maps. We use long runs of loci at which cases share a common allele identically by state to localize hypothesized predisposition genes. The distribution of such runs under the hypothesis of no genetic effect is evaluated by simulation. Methods are illustrated by analysis of an extended prostate cancer pedigree previously reported to show significant linkage to chromosome 1p23. Our analysis establishes that runs of simple single locus statistics can be powerful, tractable and robust for finding DNA shared between relatives, and that extended pedigrees offer powerful designs for gene detection based on these statistics.

Genome-wide linkage analysis for aggressive prostate cancer in Utah high-risk pedigrees.

BACKGROUND: It has been proposed that studying alternative phenotypes, such as tumor aggressiveness, may be a solution for overcoming the apparent heterogeneity that has hindered the identification of prostate cancer (PC) genes. We present the results of a genome-scan for predisposition to aggressive PC using the Utah high-risk pedigree resource. METHODS: We identified 259 subjects with aggressive PC in 57 extended and nuclear families. Parametric and non-parametric multipoint linkage statistics were calculated for a genome-wide set of 401 microsatellite markers using the MCLINK software package. Stratification analyses by the number of affected subjects per pedigree (<5, >or=5) and the average age at diagnosis of affected subjects (<70 years, >or=70 years) were also performed. RESULTS: No significant results were observed at the genome-wide level, but suggestive evidence for linkage was observed on chromosomes 9q (HLOD = 2.04) and 14q (HLOD = 2.08); several pedigrees showed individual evidence for linkage at each locus (LOD > 0.58). The subset of pedigrees with earlier age at onset demonstrated nominal linkage evidence on chromosomes 3q (HLOD = 1.79), 8q (HLOD = 1.67), and 20q (HLOD=1.82). The late-onset subset showed suggestive linkage on chromosome 6p (HLOD = 2.37) and the subset of pedigrees with fewer than five affected subjects showed suggestive linkage on chromosome 10p (HLOD = 1.99). CONCLUSIONS: Linkage evidence observed on chromosomes 6p, 8q, and 20q support previously reported PC aggressiveness loci. While these results are encouraging, further research is necessary to identify the gene or genes responsible for PC aggressiveness and surmount the overarching problem of PC heterogeneity.

Identification and study of Utah pseudo-isolate populations-prospects for gene identification.

Isolate populations of varied types have proven powerful for gene identification for rare Mendelian disorders, and continue to show such promise for more complex phenotypes. Existing isolate populations are limited in the phenotypes available for study, and new population isolates are unlikely to arise. We utilize genealogical data available for the state of Utah, dating back to its European founders, to retrospectively define and examine pseudo-isolate subpopulations. These pseudo-isolate populations are defined by selection of a set of "founders" from the genealogical data, and then limitation of "immigration" by censoring of matings and offspring that do not match the isolate population design. A wide variety of pseudo isolate and other study designs are possible by varying the number and type of founders and the extent of immigration allowed. We present several different example Birth-Country pseudo-isolate populations defined within the Utah Population Database (UPDB). We utilize linked cancer phenotype data available for the Utah population to show the utility of this pseudo-isolate approach for identification of more genetically homogeneous prostate cancer pedigrees for predisposition gene identification. In conclusion, we present a unique approach to retrospective "creation" of isolate populations using existing genealogical data. We use the UPDB to exhibit the utility of this approach for the highly heterogeneous Utah population, and suggest the approach is feasible for any population for which high quality genealogy and phenotype data are available.

Evidence of a pharmacogenomic response to interleukin-l receptor antagonist in rheumatoid arthritis.

Biological activity of the IL-1 system depends on the balance between two proinflammatory proteins (IL-1alpha and IL-1beta) and the related anti-inflammatory protein, the IL-1 receptor antagonist (IL-1Ra). The genes for these proteins lie within 430 kb on human chromosome 2. Based on a clinical trial of human recombinant IL-1ra in rheumatoid arthritis, we tested whether IL-1 genotype might be related to the likelihood of response to anti-IL-1 therapy. A positive response was defined as a reduction of at least 50% in the number of swollen joints by week 24, following treatment with either 150 mg/day IL-1ra or placebo. The response rate to treatment, independent of genotype, was 48% (44/91). A highly significant association was found between carriage of the rarer allele at IL1A(+4845) and response to treatment (P=0.0009; OR=4.85 (1.85,12.70)). The response rate in patients carrying this allele was 63.4% compared with 26.3% in noncarriers. A weaker association was found for IL1B(+3954) (P=0.02). There was a highly significant interaction between treatment (150 mg/day or placebo) and the composite genotype across IL1A(+4845) and IL1B(+3954) (P=7.6 x 10(-5)). No associations with IL-1 genotypes were found in patients receiving placebo. Thus, a significant pharmacogenomic effect was found in the treatment of RA patients with recombinant IL-1ra.

Generating genetic risk scores from intermediate phenotypes for use in association studies of clinically significant endpoints.

While previous results of genetic association studies for common, complex diseases (eg., coronary artery disease, CAD) have been disappointing, examination of multiple related genes within a physiologic pathway may provide improved resolution. This paper describes a method of calculating a genetic risk score (GRS) for a clinical endpoint by integrating data from many candidate genes and multiple intermediate phenotypes (IPs). First, the association of all single nucleotide polymorphisms (SNPs) to an IP is determined and regression beta-coefficients are used to calculate an IP-specific GRS for each individual, repeating this analysis for every IP. Next, the IPs are assessed by a second regression as predictors of the clinical endpoint. Each IP's individual GRS is then weighted by the regression beta-coefficients from the second step, creating a single, composite GRS. As an example, 3,172 patients undergoing coronary angiography were evaluated for 3 SNPs from the cholesterol metabolism pathway. Although these data provide only a preliminary example, the GRS method detected significant differences in CAD by GRS group, whereas separate genotypes did not. These results illustrate the potential of the GRS methodology for multigenic risk evaluation and suggest that such approaches deserve further examination in common, complex diseases such as CAD.

Functional correlates of the interleukin-1 receptor antagonist gene polymorphism in the colonic mucosa in ulcerative colitis.

Association studies have identified the interleukin-1 receptor antagonist gene allele 2(IL-1RN*2) as a marker of susceptibility in ulcerative colitis (UC). This study investigated the significance of the IL-1RN genotype with respect to protein and mRNA expression in the colonic mucosa. Homogenates of rectal biopsies from 99 UC and 54 controls were assayed for cytokines IL-1ra, IL-1a and IL-1b using ELISA. IL1RN, IL1A and IL1B genotypes were determined using restriction-enzyme analysis. The ability of the two IL1RN alleles to generate steady-state mRNA accumulation was assessed in the colonic mucosa of seven heterozygous patients. Stepwise linear regression demonstrated that IL-1RN genotype (P=0.001), diagnosis (P<0.0001) and treatment (P<0.03) were independent factors associated with the IL-1ra protein level whilst IL1RN genotype (P=0.005) and macroscopic inflammatory grade (P<0.0001) were associated with the IL-1ra/ total IL-1 ratio. The IL1RN*2 correlated with reduced IL-1ra and IL-1ra/IL-1 ratio with a gene dosage effect. In heterozygous UC patients the ratio of allele 1 mRNA / allele 2 steady state mRNA was always greater than 1 (range: 1.2-3.1) (P=0.018). The IL-1RN*2 is associated with reduced levels of IL-1ra protein and IL-1RN mRNA in the colonic mucosa, providing a biologically plausible explanation for the observed association of the allele with the disease.

Genomewide multipoint linkage analysis of seven extended Palauan pedigrees with schizophrenia, by a Markov-chain Monte Carlo method.

Palauans are an isolated population in Micronesia with lifetime prevalence of schizophrenia (SCZD) of 2%, compared to the world rate of approximately 1%. The possible enrichment for SCZD genes, in conjunction with the potential for reduced etiological heterogeneity and the opportunity to ascertain statistically powerful extended pedigrees, makes Palauans a population of choice for the mapping of SCZD genes. We have used a Markov-chain Monte Carlo method to perform a genomewide multipoint analysis in seven extended pedigrees from Palau. Robust multipoint parametric and nonparametric linkage (NPL) analyses were performed under three nested diagnostic classifications-core, spectrum, and broad. We observed four regions of interest across the genome. Two of these regions-on chromosomes 2p13-14 (for which, under core diagnostic classification, NPL=6.5 and parametric LOD=4.8) and 13q12-22 (for which, under broad diagnostic classification, parametric LOD=3.6, and, under spectrum diagnostic classification, parametric LOD=3.5)-had evidence for linkage with genomewide significance, after correction for multiple testing; with the current pedigree resource and genotyping, these regions are estimated to be 4.3 cM and 19.75 cM in size, respectively. A third region, with intermediate evidence for linkage, was identified on chromosome 5q22-qter (for which, under broad diagnostic classification, parametric LOD=2.5). The fourth region of interest had only borderline suggestive evidence for linkage (on 3q24-28; for this region, under broad diagnostic classification, parametric LOD=2.0). All regions exhibited evidence for genetic heterogeneity. Our findings provide significant evidence for susceptibility loci on chromosomes 2p13-14 and 13q12-22 and support both a model of genetic heterogeneity and the utility of a broader set of diagnostic classifications in the population from Palau.

Interleukin 1 receptor antagonist gene polymorphism and restenosis after coronary angioplasty.

BACKGROUND: Percutaneous transluminal coronary angioplasty (PTCA) is limited by the recurrence of luminal stenosis, which occurs in up to 50% of procedures. It has been shown that patient specific factors, perhaps genes, contribute to this process. OBJECTIVE: To determine whether completion of healing after PTCA is part of an acute self limiting inflammatory process and whether polymorphism at important inflammatory gene loci might determine susceptibility to restenosis after PTCA. DESIGN: DNA samples were collected from 171 patients attending for elective PTCA in Sheffield (S) and Leicester (L), who were scheduled to undergo follow up angiography (at four months (L) or six months (S)) as part of other restenosis studies. At follow up angiography, the patients were separated into restenosers (> 50% luminal narrowing) and non-restenosers (< 50% luminal narrowing). Four DNA polymorphisms within interleukin 1 (IL-1) related loci (IL-1A (-889), IL-1B (-511), IL-1B (+3954), and IL-1RN intron 2 VNTR (variable number tandem repeat)) were genotyped using methods based on polymerase chain reaction. Significance was assessed by chi(2) analysis of the relevant contingency table, and the magnitude of effect was estimated by calculating odds ratios. The Mantel-Haenszel (MH) test was applied to summarise data across the two populations. RESULTS: Allele 2 at IL-1RN (IL-1RN*2) was significantly over represented in the non-restenoser group (L+S, 34% v 23% in restenosers). Furthermore, IL-1RN*2 homozygosity was increased in the non-restenoser population compared with the restenosers (MH test: p = 0.0196 (L+S); p = 0.031 (L+S, single vessel disease only), and the effect seemed to be restricted to the single vessel disease subpopulation. For other polymorphism within IL-1 related loci no significant associations were found with either restenosis or non-restenosis. CONCLUSIONS: IL-1RN*2 may be associated with protection from restenosis after PTCA for individuals with single vessel disease. As this polymorphism has functional significance, this finding suggests that alteration in an individual's inflammatory predisposition may modulate the blood vessel response to injury.

Association of the interleukin 1 receptor antagonist gene with ulcerative colitis in Northern European Caucasians.

BACKGROUND AND AIMS: An association between the allele 2 of the interleukin 1 receptor antagonist gene variable number tandem repeats polymorphism in intron 2 and ulcerative colitis was first reported in 1994. Subsequent studies in Caucasian Northern European patients have not confirmed this, although trends towards an association were observed. The lack of statistical significance could reflect inadequate power. In this study the association was reassessed in a large independent set of well characterised Caucasian patients and a meta-analysis of reported patient series was performed. PATIENTS AND METHODS: A total of 320 patients with endoscopically and histologically confirmed ulcerative colitis (124 pancolitis, 196 left sided and distal disease) and 827 ethnically matched controls were genotyped at polymorphic sites in the interleukin 1 receptor antagonist gene. Carriage rates were compared using chi(2) statistics. A meta-analysis of this and seven previous studies in North European Caucasian patients was performed using the Mantel-Haenszel chi(2) test. RESULTS: Patients had a significantly increased carriage rate of allele 2 compared with controls (52% v 45%; odds ratio 1.3 (95% confidence interval (CI) 1.01-1.7); p=0.04). The allele 2 carriage rate was highest in extensive colitis (carriage rate 56%; odds ratio 1.5 (95% CI 1.1-2.3) p=0.02) and in individuals who had undergone colectomy (carriage rate 55%; odds ratio 1.5 (95% CI 0.95-2.4); p=0.08). Meta-analysis of all eight studies showed a significant association between carriage of allele 2 and ulcerative colitis (odds ratio 1.23 (95% CI 1.04-1.45); p=0.01). CONCLUSIONS: The association of the interleukin 1 receptor antagonist gene polymorphism with ulcerative colitis is confirmed. The association is minor and confers only a small risk to an individual but will contribute a high attributable risk in a population due to the high allelic frequency. Accurate phenotypic characterisation defines more homogeneous subsets of patients, such as those with extensive disease, in whom the association is greater.

A candidate prostate cancer susceptibility gene at chromosome 17p.

It is difficult to identify genes that predispose to prostate cancer due to late age at diagnosis, presence of phenocopies within high-risk pedigrees and genetic complexity. A genome-wide scan of large, high-risk pedigrees from Utah has provided evidence for linkage to a locus on chromosome 17p. We carried out positional cloning and mutation screening within the refined interval, identifying a gene, ELAC2, harboring mutations (including a frameshift and a nonconservative missense change) that segregate with prostate cancer in two pedigrees. In addition, two common missense variants in the gene are associated with the occurrence of prostate cancer. ELAC2 is a member of an uncharacterized gene family predicted to encode a metal-dependent hydrolase domain that is conserved among eukaryotes, archaebacteria and eubacteria. The gene product bears amino acid sequence similarity to two better understood protein families, namely the PSO2 (SNM1) DNA interstrand crosslink repair proteins and the 73-kD subunit of mRNA 3' end cleavage and polyadenylation specificity factor (CPSF73).

A new nonparametric linkage statistic for mapping both qualitative and quantitative trait loci.

We describe an alternative nonparametric linkage (NPL) statistic to that of Kruglyak et al. [Am. J. Hum. Genet. 58:1347-63, 1996] that can be used with qualitative phenotypes, and is easily extended for use with quantitative phenotypes. We analyzed the Genetic Analysis Workshop 12 simulated isolated population data, replicate 1, using two phenotypes; affected status (AFF) a dichotomous phenotype and quantitative trait Q5, which was chosen since it was the most strongly associated with AFF. One false positive significant NPL score was observed for the AFF phenotype. For Q5 a single region on chromosome 1 reached genome-wide significance. The peak of this signal was for marker D01G137 at 135.1 cM with a quantitative trait locus (QTL)-NPL score of 4.19. The nearest marker to the true location of the major gene (MG5 at 137.1 cM) was D01G139 at position 135.8 cM, where the QTL-NPL score was still high at 4.08.

A robust multipoint linkage statistic (tlod) for mapping complex trait loci.

Classical parametric two-point linkage analysis is a powerful analysis tool, however there are clear disadvantages too, including the sensitivity to allele frequency mis-specification. Conversely, multipoint linkage analysis is not sensitive to allele frequency mis-specification, but it is sensitive to genetic model mis-specification. Göring and Terwilliger [Am J Hum Genet 66:1095-106, 2000] proposed a new robust multipoint statistic that increased the robustness of multipoint analyses. In this paper we have referred to this new statistic as the tlod. We applied this new statistic to the Genetic Analysis Workshop (GAW) 12 data using affected status (AFF) as the phenotype of interest. The heterogeneity tlod and two-point hlod scores correlated highly across the genome (p < 0.0001), as expected, but the het-tlod had a lower number false positives. In addition, the tlod analysis handled missing data better, as would be expected for a multipoint method. When one-third of the genotype data was removed (dead people) the tlod analysis was less affected than the two-point analysis. When tlod scores were compared with multipoint lod scores in true gene locations, the robustness of the tlod to model mis-specification was clearly evident. When the "best" replicate from the general population was analyzed, a borderline genome-wide significant two-point hlod result (3.6) was found 4 cM from MG6 and MG7 on chromosome 6. The heterogeneity tlod score was lower than the two-point hlod score (1.8), but greater than the heterogeneity multipoint lod score (0.4). However, when replicate 1 of the isolated population was analyzed none of the true gene locations were identified with either statistic.

Allelic association in large pedigrees.

We subjected the first replication of the simulated isolated population data set to a novel analysis for association between marker alleles and either disease phenotypes or quantitative variable. The analysis depends on being able to reliably reconstruct all haplotypes in the pedigree. This was achieved using the MCLINK blocked Gibbs sampling program. We observed a highly significant association between the variable Q5 and marker D01G138, and suggestive associations between the disease trait and markers D03G056 and D07G004.

Correcting for multiple analyses in genomewide linkage studies.

The dissection of complex traits frequently calls for multiple analyses to be performed, including the use of both multiple phenotypes and genetic models. These multiple phenotypes and models are often not independent, and hence the necessary correction for the multiple testing is not straightforward. In this paper we offer a new approach to address the problem of how to correct for non-independent multiple analyses in genomewide linkage studies. We describe one method of how to determine the number of 'effectively independent' tests performed in a linkage study using simple linear regression techniques. Further we describe how to use such information to establish genomewide significance thresholds for infinitely dense genomewide maps.

An interleukin-1 genotype is associated with fatal outcome of meningococcal disease.

To determine whether known variants of the interleukin-1 (IL-1) and tumor necrosis factor (TNF) gene families are associated with severe manifestations of meningococcal disease, 276 white patients 4-70 years of age (median, 17 years) were genotyped. All patients had microbiologically proven Neisseria meningitidis infection; 39 died and 237 survived. A significant association (P<.001) was found between fatal outcome and genotype at IL1B (nucleotide position -511). Homozygous individuals, both for the common (1/1) and the rare (2/2) alleles, had increased odds ratios (ORs) for death, compared with heterozygous individuals (1/2): ORs (95% confidence intervals [CIs]) were 3.39 (1.39-8.29) and 7.35 (2.51-21.45), respectively. The mortality rates according to genotype at IL1B (-511) were 18.0% (1/1), 6.1% (1/2), and 32.3% (2/2), compared with 14.2% overall. The composite genotype, consisting of heterozygosity of IL1B (-511) together with homozygosity of the common allele of the IL-1 receptor antagonist gene (IL1RN) at +2018, was significantly associated with survival (P=.018; OR, 7.78 [95% CI, 1. 05-59.05]). There was no association between TNF genotype and fatal outcome. These data suggest that IL-1 genotype influences the severity of meningococcal disease.