Significant Linkage Peak Research Articles

Abstract 137: Identification Of A Noncoding Genetic Variant In The Tissue Factor Locus That Reverses Lethal Thrombosis In Mice

Background: Thrombosis is initiated by tissue factor (TF, gene name F3 ) binding to coagulationFVII, with tissue factor pathway inhibitor (TFPI) inhibiting this complex. Alterations in TF orTFPI expression significantly affect thrombosis. Reducing TFPI expression by 50% ( Tfpi +/-) inmice results in a perinatal lethal phenotype on the Factor V Leiden homozygous( F5 L/L )prothrombotic background. We used the F5 L/L Tfpi +/- lethal phenotype to conduct a dominantsensitized whole genome ENU mutagenesis screen to suppress the F5 L/L Tfpi +/- lethality. Weidentified a Modifier of Factor 5 Leiden 6 (MF5L6) line with 72% penetrance and 85 F5 L/L Tfpi +/- offspring. A significant linkage peak (LOD=4.35),explaining half the suppressing effect andcontaining F3 (Chromosome 3) was identified. Goals/Hypothesis: To identify the genomic variant controlling F3 expression in the MF5L6 line. Methods: To quantify F3 expression in the surviving mice from MF5L6, quantitative PCR onliver, lung, and heart tissues from MF5L6 was performed. We used Sanger DNA and highthroughput sequencing to identify candidate TF regulatory variants in the F3 locus. Theprothrombin time assay was used to test the effects of reduced TF expression on in vitro bloodcoagulation. Results: Two distinct expression profiles in the lung and liver of the MF5L6 mice wereobserved, those that had a 50% reduction in F3 mRNA and those that did not. Heart tissuesexhibited one expression profile, suggesting that the variant regulates F3 expressiontissue-specifically. Sanger sequencing of the F3 coding region revealed no coding mutations inMF5L6 mice. Whole genome sequencing identified two novel candidate variants (in unknownF3 regulatory elements) in the 200 kilobase upstream region of F3 . The 50% reduction in F3 resulted in significant changes in coagulation by the prothrombin assay (n=18,p<0.0009). Conclusion: We identified novel candidate variants for regulating F3 gene expression and aredetermining their mechanism of action. Investigation of these variants will provide new insightsinto the regulation of F3 and enable us to identify the variant(s) responsible for the remainder ofthe thrombosis suppressing effect in MF5L6. Our findings provide new insights into the geneticregulation of thrombosis.

Abstract 2497: Inherited copy number variants that increase risk of developing pediatric germ cell tumors with a particular focus on the X chromosome

Abstract Pediatric malignant germ cell tumors (GCTs) are heterogeneous but are grouped together due to the presumed common cell of origin, the primordial germ cell (PGC). Little is known about etiology; however, evidence supporting in utero origins of pediatric and adult GCT suggests that disruptions in normal germ cell development are likely to be highly relevant. Germline copy number variants (CNVs) are a plausible source of inherited genetic variation and have not been thoroughly evaluated to date. CNVs on the X chromosome are of particular interest because individuals with both Klinefelter syndrome (47, XXY) and Turner syndrome (45, X) are at increased risk of pediatric GCT, suggesting that alterations in X chromosome dosage contribute to etiology. A family study of adult testicular cancer where at least one member had bilateral cancer identified a significant linkage peak at Xq27 (hLOD=4.7). In addition, somatic copy number gains of chromosome X are frequently seen in adult testicular germ cell tumors. Given these findings in adult GCT, similar aberrations may also be relevant risk factors for the pediatric group. Pediatric GCT samples were obtained from a Children’s Oncology Group study and state biobank programs in Michigan and California. Genotyping array data were generated using the Illumina HumanCoreExome Beadchip (Illumina, San Diego). CNV calls were made for 2,002 GCT cases and 1,402 controls with high quality intensity data using Genvisis, which has specialized algorithms to make CNV calls on the X chromosome. Klinefelter syndrome cases (n=27) were excluded from the analyses. No study sample had classic Turner syndrome; however, one female case had a giant 51Mb deletion of Xq. Analyses were performed for females-only, males-only, and everyone together. We identified two regions that are study-wide significant on chromosome X at Xq27.1 and Xp11.2 and one nominally significant near Xq28. The most significant finding was identified in females-only and is located within a region that encodes genes within the SPANX family. Evidence suggests these genes affect germ cells; however, supporting data is reported in spermatozoa rather than oocytes. The events in our study were large (240kb), had similar breakpoints, and appear to be flanked by repetitive DNA that might lead to recurrent events at the same region. The variant was present in the control samples within gnomAD at a low allele frequency (0.18%). In our study, both deletions and duplications were noted: 11 female cases (1 or 2 expected based on gnomAD allele frequencies) and 0 female controls (0-1 expected). The finding was not replicated in males (6 male cases and 5 male controls harbored similar CNVs). We are in the process of validating these in silico and potentially via quantitative PCR. Findings may shed light on disruptions in normal development that may increase risk of GCT. Citation Format: Shannon S. Cigan, John J. Meredith, John Zaharick, Erica Langer, Anthony J. Hooten, John A. Lane, Nathan Pankratz, Jenny N. Poynter. Inherited copy number variants that increase risk of developing pediatric germ cell tumors with a particular focus on the X chromosome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2497.

Identification of Chromosomal Regions Linked to Autism-Spectrum Disorders: A Meta-Analysis of Genome-Wide Linkage Scans.

Background: Autism spectrum disorder (ASD) is a clinically and genetically heterogeneous group of pervasive neurodevelopmental disorders with a strong hereditary component. Although, genome-wide linkage scans (GWLS) and association studies (GWAS) have previously identified hundreds of ASD risk gene loci, the results remain inconclusive. Method: We performed a heterogeneity-based genome search meta-analysis (HEGESMA) of 15 genome scans of autism and ASD. Results: For strictly defined autism, data were analyzed across six separate genome scans. Region 7q22-q34 reached statistical significance in both weighted and unweighted analyses, with evidence of significantly low between-scan heterogeneity. For ASDs (data from 12 separate scans), chromosomal regions 5p15.33-5p15.1 and 15q22.32-15q26.1 reached significance in both weighted and unweighted analyses but did not reach significance for either low or high heterogeneity. Region 1q23.2-1q31.1 was significant in unweighted analyses with low between-scan heterogeneity. Finally, region 8p21.1-8q13.2 reached significant linkage peak in all our meta-analyses. When we combined all available genome scans (15), the same results were produced. Conclusions: This meta-analysis suggests that these regions should be further investigated for autism susceptibility genes, with the caveat that autism spectrum disorders have different linkage signals across genome scans, possibly because of the high genetic heterogeneity of the disease.

Myopia in African Americans Is Significantly Linked to Chromosome 7p15.2-14.2.

PurposeThe purpose of this study was to perform genetic linkage analysis and association analysis on exome genotyping from highly aggregated African American families with nonpathogenic myopia. African Americans are a particularly understudied population with respect to myopia.MethodsOne hundred six African American families from the Philadelphia area with a family history of myopia were genotyped using an Illumina ExomePlus array and merged with previous microsatellite data. Myopia was initially measured in mean spherical equivalent (MSE) and converted to a binary phenotype where individuals were identified as affected, unaffected, or unknown. Parametric linkage analysis was performed on both individual variants (single-nucleotide polymorphisms [SNPs] and microsatellites) as well as gene-based markers. Family-based association analysis and transmission disequilibrium test (TDT) analysis modified for rare variants was also performed.ResultsGenetic linkage analysis identified 2 genomewide significant variants at 7p15.2 and 7p14.2 (in the intergenic region between MIR148A and NFE2L3 and in the noncoding RNA LOC401324) and 2 genomewide significant genes (CRHR2 and AVL9) both at 7p14.3. No genomewide results were found in the association analyses.ConclusionsThis study identified a significant linkage peak in African American families for myopia at 7p15.2 to 7p14.2, the first potential risk locus for myopia in African Americans. Interesting candidate genes are located in the region, including PDE1C, which is highly expressed in the eyes, and known to be involved in retinal development. Further identification of the causal variants at this linkage peak will help elucidate the genetics of myopia in this understudied population.

A linkage and exome study of multiplex families with bipolar disorder implicates rare coding variants of ANK3 and additional rare alleles at 10q11-q21.

BackgroundBipolar disorder is a highly heritable psychiatric condition for which specific genetic factors remain largely unknown. In the present study, we used combined whole-exome sequencing and linkage analysis to identify risk loci and dissect the contribution of common and rare variants in families with a high density of illness.MethodsOverall, 117 participants from 15 Australian extended families with bipolar disorder (72 with affective disorder, including 50 with bipolar disorder type I or II, 13 with schizoaffective disorder–manic type and 9 with recurrent unipolar disorder) underwent whole-exome sequencing. We performed genome-wide linkage analysis using MERLIN and conditional linkage analysis using LAMP. We assessed the contribution of potentially functional rare variants using a gene-based segregation test.ResultsWe identified a significant linkage peak on chromosome 10q11-q21 (maximal single nucleotide polymorphism = rs10761725; exponential logarithm of the odds [LODexp] = 3.03; empirical p = 0.046). The linkage interval spanned 36 protein-coding genes, including a gene associated with bipolar disorder, ankyrin 3 (ANK3). Conditional linkage analysis showed that common ANK3 risk variants previously identified in genome-wide association studies — or variants in linkage disequilibrium with those variants — did not explain the linkage signal (rs10994397 LOD = 0.63; rs9804190 LOD = 0.04). A family-based segregation test with 34 rare variants from 14 genes under the linkage interval suggested rare variant contributions of 3 brain-expressed genes: NRBF2 (p = 0.005), PCDH15 (p = 0.002) and ANK3 (p = 0.014).LimitationsWe did not examine non-coding variants, but they may explain the remaining linkage signal.ConclusionCombining family-based linkage analysis with next-generation sequencing data is effective for identifying putative disease genes and specific risk variants in complex disorders. We identified rare missense variants in ANK3, PCDH15 and NRBF2 that could confer disease risk, providing valuable targets for functional characterization.

Linkage of Alzheimer disease families with Puerto Rican ancestry identifies a chromosome 9 locus

The genetic admixture of Caribbean Hispanics provides an opportunity to discover novel genetic factors in Alzheimer disease (AD). We sought to identify genetic variants for AD through a family-based design using the Puerto Rican (PR) Alzheimer Disease Initiative (PRADI). Whole-genome sequencing (WGS) and parametric linkage analysis were performed for 100 individuals from 23 multiplex PRADI families. Variants were prioritized by minor allele frequency (<0.01), functional potential [combined annotation dependent depletion score (CADD) >10], and co-segregation with AD. Variants were further ranked using an independent PR case-control WGS dataset (PR10/66). A genome-wide significant linkage peak was found in 9p21 with a heterogeneity logarithm of the odds score (HLOD) >5.1, which overlaps with an AD linkage region from two published independent studies. The region harbors C9orf72, but no expanded repeats were observed in the families. Seven variants prioritized by the PRADI families also displayed evidence for association in the PR10/66 (p < 0.05), including a missense variant in UNC13B. Our study demonstrated the importance of family-based design and WGS in genetic study of AD.

Identification of a Novel Candidate Gene for Serrated Polyposis Syndrome Germline Predisposition by Performing Linkage Analysis Combined With Whole-Exome Sequencing.

OBJECTIVES:Serrated polyposis syndrome (SPS) is a complex disorder with a high risk of colorectal cancer for which the germline factors remain largely unknown. Here, we combined whole-exome sequencing (WES) and linkage studies in families with multiple members affected by SPS to identify candidate genes harboring rare variants with higher penetrance effects.METHODS:Thirty-nine affected subjects from 16 extended SPS families underwent WES. Genome-wide linkage analysis was performed under linear and exponential models. The contribution of rare coding variants selected to be highly pathogenic was assessed using the gene-based segregation test.RESULTS:A significant linkage peak was identified on chromosome 3p25.2-p22.3 (maxSNP = rs2293787; LODlinear = 2.311, LODexp = 2.11), which logarithm of the odds (LOD) score increased after fine mapping for the same marker (maxSNP = rs2293787; LODlinear = 2.4, LODexp = 2.25). This linkage signal was replicated in 10 independent sets of random markers from this locus. To assess the contribution of rare variants predicted to be pathogenic, we performed a family-based segregation test with 11 rare variants predicted to be deleterious from 10 genes under the linkage intervals. This analysis showed significant segregation of rare variants with SPS in CAPT7, TMEM43, NGLY1, and FBLN2 genes (weighted P value > 0.007).DISCUSSION:Protein network analysis suggested FBLN2 as the most plausible candidate genes for germline SPS predisposition. Etiologic rare variants implicated in disease predisposition may be identified by combining traditional linkage with WES data. This powerful approach was effective for the identification of a new candidate gene for hereditary SPS.

Rare variants and loci for age-related macular degeneration in the Ohio and Indiana Amish

Age-related macular degeneration (AMD) is a leading cause of blindness in the world. While dozens of independent genomic variants are associated with AMD, about one-third of AMD heritability is still unexplained. To identify novel variants and loci for AMD, we analyzed Illumina HumanExome chip data from 87 Amish individuals with early or late AMD, 79 unaffected Amish individuals, and 15 related Amish individuals with unknown AMD affection status. We retained 37,428 polymorphic autosomal variants across 175 samples for association and linkage analyses. After correcting for multiple testing (n = 37,428), we identified four variants significantly associated with AMD: rs200437673 (LCN9, p = 1.50 × 10−11), rs151214675 (RTEL1, p = 3.18 × 10−8), rs140250387 (DLGAP1, p = 4.49 × 10−7), and rs115333865 (CGRRF1, p = 1.05 × 10−6). These variants have not been previously associated with AMD and are not in linkage disequilibrium with the 52 known AMD-associated variants reported by the International AMD Genomics Consortium based on physical distance. Genome-wide significant linkage peaks were observed on chromosomes 8q21.11–q21.13 (maximum recessive HLOD = 4.03) and 18q21.2–21.32 (maximum dominant HLOD = 3.87; maximum recessive HLOD = 4.27). These loci do not overlap with loci previously linked to AMD. Through gene ontology enrichment analysis with ClueGO in Cytoscape, we determined that several genes in the 1-HLOD support interval of the chromosome 8 locus are involved in fatty acid binding and triglyceride catabolic processes, and the 1-HLOD support interval of the linkage region on chromosome 18 is enriched in genes that participate in serine-type endopeptidase inhibitor activity and the positive regulation of epithelial to mesenchymal transition. These results nominate novel variants and loci for AMD that require further investigation.

T55 - The Genetic Basis Of The Comorbidity Between Cannabis Use And Major Depression

While the prevalence of major depression is known to be elevated amongst cannabis users, the causes of this comorbidity are not clear. Here we investigate the role of genetics in this relationship and identify genomic loci linked to these traits. Using a sample of Mexican American extended families (n=1,284), we use variance decomposition methods to establish the degree of genetic correlation between cannabis use and major depression. Genome-wide univariate and bivariate linkage scans are conducted to localize the chromosomal regions influencing these traits and the comorbidity observed between them. Both major depression (h2=0.349, p=1.06x10-5, SE=0.100) and cannabis use (h2=0.614, p=1.00x10-6, SE=0.151) are heritable traits, and there is significant genetic correlation between the two (ρg=0.424, p=0.0364, SE=0.195). Genome-wide linkage scans identify a significant univariate linkage peak for major depression on chromosome 22 (LOD=3.144 at 2cM), with a suggestive peak for cannabis use on chromosome 21 (LOD=2.123 at 37cM). A significant pleiotropic linkage peak influencing both major depression and cannabis use was identified on chromosome 11, using a bivariate model (LOD=3.229 at 112cM). This location spans the NCAM1-TTC12-ANKK1-DRDR2 gene cluster. Follow-up of this pleiotropic signal provided tentative evidence implicating a rare SNP 20kb upstream of NCAM1 (s7932341); with peak-wide significant bivariate association with cannabis use and major depression (p=3.10x10-5). We show that genetic influences play an important role in the comorbidity between cannabis use and major depression.Specifically, we identify a pleiotropic locus on chromosome 11, spanning the NCAM1-TTC12-ANKK1-DRDR2 gene cluster, which has been previously implicated in both addiction and depression research.

The genetic basis of the comorbidity between cannabis use and major depression.

While the prevalence of major depression is elevated among cannabis users, the role of genetics in this pattern of comorbidity is not clear. This study aimed to estimate the heritability of cannabis use and major depression, quantify the genetic overlap between these two traits and localize regions of the genome that segregate in families with cannabis use and major depression. Family-based univariate and bivariate genetic analysis. San Antonio, Texas, USA. Genetics of Brain Structure and Function study (GOBS) participants: 1284 Mexican Americans from 75 large multi-generation families and an additional 57 genetically unrelated spouses. Phenotypes of life-time history of cannabis use and major depression, measured using the semistructured MINI-Plus interview. Genotypes measured using ~1M single nucleotide polymorphisms (SNPs) on Illumina BeadChips. A subselection of these SNPs were used to build multi-point identity-by-descent matrices for linkage analysis. Both cannabis use [h2 =0.614, P=1.00×10-6 , standard error (SE)=0.151] and major depression (h2 =0.349, P=1.06×10-5 , SE=0.100) are heritable traits, and there is significant genetic correlation between the two (ρg =0.424, P=0.0364, SE=0.195). Genome-wide linkage scans identify a significant univariate linkage peak for major depression on chromosome 22 [logarithm of the odds (LOD)=3.144 at 2centimorgans (cM)], with a suggestive peak for cannabis use on chromosome 21 (LOD=2.123 at 37cM). A significant pleiotropic linkage peak influencing both cannabis use and major depression was identified on chromosome 11 using a bivariate model (LOD=3.229 at 112cM). Follow-up of this pleiotropic signal identified a SNP 20kb upstream of NCAM1 (rs7932341) that shows significant bivariate association (P=3.10×10-5 ). However, this SNP is rare (seven minor allele carriers) and does not drive the linkage signal observed. There appears to be a significant genetic overlap between cannabis use and major depression among Mexican Americans, a pleiotropy that appears to be localized to a region on chromosome 11q23 that has been linked previously to these phenotypes.

Abstract 1487: Gene expression and linkage analysis implicates CBLB as a mediator of rituximab resistance

Abstract Drug resistance remains one of the largest challenges in the curative treatment of cancer. We investigated the problem of monoclonal antibody resistance in vitro in a high throughput, complement-dependent cytotoxic (CDC) assay using immortalized as well as cancerous cell lines. First, the heritability of rituximab and ofatumumab sensitivity was explored using Epstein Barr Virus-immortalized human lymphoblastoid cell lines from two independent sources: a collection of trios (95 samples, 13 trios) from the Centre d’Etude du Polymorphisme Humain and an unrelated collection (486 samples) from the Children's Hospital Oakland Research Initiative. Cell viability in the presence or absence of 10ug/ml rituximab or 10ug/ml ofatumumab treatment was quantitated using AlamarBlue. Heritability was estimated using variance component analysis using MERLIN: H2 = 33.11% (p&amp;lt;0.001) and H2 = 31.11% (p&amp;lt;0.001) for rituximab and ofatumumab, resp. Next, public information on genotype and gene expression was used to analyze associations between loci/genes and drug response with three methods: genome-wide association, linkage, and gene expression analysis. GWAS were performed on the 486 unrelated samples: (a) rituximab responses, (b) ofatumumab responses, and (c) the vector of both responses modeled jointly. There was one suggestive (-log10(P)&amp;gt;6) result across all three analyses; this result for rituximab response occurred within the gene, SMOC2. Next, linkage analysis on the 95 related samples revealed one significant linkage peak on chromosome 12 for rituximab and two significant peaks for ofatumumab, on chromosome 12 and 3. Also, we identified genes whose mRNA expression level was correlated with the degree of either rituximab or ofatumumab sensitivity. Quantitative Significance Analysis of Microarrays revealed 13 genes whose expression was correlated with rituximab sensitivity and 25 genes whose expression was correlated with ofatumumab sensitivity (false discovery rate cutoff&amp;lt;0.001%). CBLB, present on both gene lists, was the only gene that was located in either the chromosome 3 or chromosome 12 linkage peak. As such, CBLB was chosen for further functional validation. We used RNA silencing in several cell line and cancerous models to functionally validate the role of CBLB expression on rituximab resistance. Knockdown of CBLB in cell lines, both LCLs and lymphoma, led to increased resistant to both rituximab and ofatumumab. Additionally, we performed immunofluorescence in CBLB knockdown cells. CD20 localization was altered in cells with CBLB knockdown. Our current work has uncovered new mechanistic insight into the relationship between anti-CD20 antibody susceptibility, CBLB, and CD20, illustrating the power of comprehensive genetic analyses to discover a previously unknown mediator of rituxumab response. Additional studies are ongoing to further elucidate the mechanisms of CBLB-mediated resistance as well as its clinical relevance. Citation Format: John Jack, George Small, Tammy Havener, Chad Brown, Howard McLeod, Alison Motsinger, Kristy L. Richards. Gene expression and linkage analysis implicates CBLB as a mediator of rituximab resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1487.

RTTN Mutations Cause Primary Microcephaly and Primordial Dwarfism in Humans

Primary microcephaly is a developmental brain anomaly that results from defective proliferation of neuroprogenitors in the germinal periventricular zone. More than a dozen genes are known to be mutated in autosomal-recessive primary microcephaly in isolation or in association with a more generalized growth deficiency (microcephalic primordial dwarfism), but the genetic heterogeneity is probably more extensive. In a research protocol involving autozygome mapping and exome sequencing, we recruited a multiplex consanguineous family who is affected by severe microcephalic primordial dwarfism and tested negative on clinical exome sequencing. Two candidate autozygous intervals were identified, and the second round of exome sequencing revealed a single intronic variant therein (c.2885+8A>G [p.Ser963(∗)] in RTTN exon 23). RT-PCR confirmed that this change creates a cryptic splice donor and thus causes retention of the intervening 7 bp of the intron and leads to premature truncation. On the basis of this finding, we reanalyzed the exome file of a second consanguineous family affected by a similar phenotype and identified another homozygous change in RTTN as the likely causal mutation. Combined linkage analysis of the two families confirmed that RTTN maps to the only significant linkage peak. Finally, through international collaboration, a Canadian multiplex family affected by microcephalic primordial dwarfism and biallelic mutation of RTTN was identified. Our results expand the phenotype of RTTN-related disorders, hitherto limited to polymicrogyria, to include microcephalic primordial dwarfism with a complex brain phenotype involving simplified gyration.

Genome‐wide significant linkage of schizophrenia‐related neuroanatomical trait to 12q24

The insula and medial prefrontal cortex (mPFC) share functional, histological, transcriptional, and developmental characteristics, and they serve higher cognitive functions of theoretical relevance to schizophrenia and related disorders. Meta-analyses and multivariate analysis of structural magnetic resonance imaging (MRI) scans indicate that gray matter density and volume reductions in schizophrenia are the most consistent and pronounced in a network primarily composed of the insula and mPFC. We used source-based morphometry, a multivariate technique optimized for structural MRI, in a large sample of randomly ascertained pedigrees (N = 887) to derive an insula-mPFC component and to investigate its genetic determinants. Firstly, we replicated the insula-mPFC gray matter component as an independent source of gray matter variation in the general population, and verified its relevance to schizophrenia in an independent case-control sample. Secondly, we showed that the neuroanatomical variation defined by this component is largely determined by additive genetic variation (h(2) = 0.59), and genome-wide linkage analysis resulted in a significant linkage peak at 12q24 (LOD = 3.76). This region has been of significant interest to psychiatric genetics as it contains the Darier's disease locus and other proposed susceptibility genes (e.g., DAO, NOS1), and it has been linked to affective disorders and schizophrenia in multiple populations. Thus, in conjunction with previous clinical studies, our data imply that one or more psychiatric risk variants at 12q24 are co-inherited with reductions in mPFC and insula gray matter concentration. © 2015 Wiley Periodicals, Inc.

Non-coding genomic regions possessing enhancer and silencer potential are associated with healthy aging and exceptional survival.

We have completed a genome-wide linkage scan for healthy aging using data collected from a family study, followed by fine-mapping by association in a separate population, the first such attempt reported. The family cohort consisted of parents of age 90 or above and their children ranging in age from 50 to 80. As a quantitative measure of healthy aging, we used a frailty index, called FI34, based on 34 health and function variables. The linkage scan found a single significant linkage peak on chromosome 12. Using an independent cohort of unrelated nonagenarians, we carried out a fine-scale association mapping of the region suggestive of linkage and identified three sites associated with healthy aging. These healthy-aging sites (HASs) are located in intergenic regions at 12q13-14. HAS-1 has been previously associated with multiple diseases, and an enhancer was recently mapped and experimentally validated within the site. HAS-2 is a previously uncharacterized site possessing genomic features suggestive of enhancer activity. HAS-3 contains features associated with Polycomb repression. The HASs also contain variants associated with exceptional longevity, based on a separate analysis. Our results provide insight into functional genomic networks involving non-coding regulatory elements that are involved in healthy aging and longevity.

A cautionary note on ignoring polygenic background when mapping quantitative trait loci via recombinant congenic strains.

In gene mapping, it is common to test for association between the phenotype and the genotype at a large number of loci, i.e., the same response variable is used repeatedly to test a large number of non-independent and non-nested hypotheses. In many of these genetic problems, the underlying model is a mixed model consistent of one or very few major genes concurrently with a genetic background effect, usually thought as of polygenic nature and, consequently, modeled through a random effects term with a well-defined covariance structure dependent upon the kinship between individuals. Either because the interest lies only on the major genes or to simplify the analysis, it is habitual to drop the random effects term and use a simple linear regression model, sometimes complemented with testing via resampling as an attempt to minimize the consequences of this practice. Here, it is shown that dropping the random effects term has not only extreme negative effects on the control of the type I error rate, but it is also unlikely to be fixed by resampling because, whenever the mixed model is correct, this practice does not allow to meet some basic requirements of resampling in a gene mapping context. Furthermore, simulations show that the type I error rates when the random term is ignored can be unacceptably high. As an alternative, this paper introduces a new bootstrap procedure to handle the specific case of mapping by using recombinant congenic strains under a linear mixed model. A simulation study showed that the type I error rates of the proposed procedure are very close to the nominal ones, although they tend to be slightly inflated for larger values of the random effects variance. Overall, this paper illustrates the extent of the adverse consequences of ignoring random effects term due to polygenic factors while testing for genetic linkage and warns us of potential modeling issues whenever simple linear regression for a major gene yields multiple significant linkage peaks.

Genomic View of Bipolar Disorder Revealed by Whole Genome Sequencing in a Genetic Isolate

Bipolar disorder is a common, heritable mental illness characterized by recurrent episodes of mania and depression. Despite considerable effort to elucidate the genetic underpinnings of bipolar disorder, causative genetic risk factors remain elusive. We conducted a comprehensive genomic analysis of bipolar disorder in a large Old Order Amish pedigree. Microsatellite genotypes and high-density SNP-array genotypes of 388 family members were combined with whole genome sequence data for 50 of these subjects, comprising 18 parent-child trios. This study design permitted evaluation of candidate variants within the context of haplotype structure by resolving the phase in sequenced parent-child trios and by imputation of variants into multiple unsequenced siblings. Non-parametric and parametric linkage analysis of the entire pedigree as well as on smaller clusters of families identified several nominally significant linkage peaks, each of which included dozens of predicted deleterious variants. Close inspection of exonic and regulatory variants in genes under the linkage peaks using family-based association tests revealed additional credible candidate genes for functional studies and further replication in population-based cohorts. However, despite the in-depth genomic characterization of this unique, large and multigenerational pedigree from a genetic isolate, there was no convergence of evidence implicating a particular set of risk loci or common pathways. The striking haplotype and locus heterogeneity we observed has profound implications for the design of studies of bipolar and other related disorders.

Abstract 1341: Identification of significant linkage evidence for lethal prostate cancer on chromosome arm 11p15.

Abstract We performed genome wide linkage analysis in a set of high-risk prostate cancer pedigrees, each with 3 or more sampled cases whose death certificate indicated a contribution of prostate cancer to death (lethal prostate cancer). All prostate cancer cases were present in the Utah SEER Cancer Registry (pathology confirmed); all “lethal” prostate cancer cases were also required to have a Utah death certificate indicating prostate cancer as a contributing cause of death. A set of markers with no linkage disequilibrium was selected for linkage analysis. We took the intersection of SNP markers from five Illumina genotyping platforms used (550K, 610K, 1M,Omni_express (720k), and Omni_1M), resulting in selection of 301,646 markers. We began with the first marker on each chromosome and then skipped markers until one satisfied all criteria (min 0.1 cM distance, heterozygosity 0.35, R-squared &amp;lt; 0.16 -using HapMap's LD files based on CEPH). This resulted in selection of a set of 25,436 SNP LD-free genome wide markers for linkage. We have performed genome-wide linkage analysis for 21 of the genotyped high-risk lethal prostate cancer pedigrees with the 25,436 selected SNP markers. Genome wide hetLODs showed one significant linkage peak for the recessive model on chromosome 14 (LOD = 4.20). The pedigree with the highest LOD in this region has a +2.69; we are attempting to expand sampling for other pedigrees with linkage evidence in this region. A single pedigree with 16 prostate cancer cases, at least 4 of whom have already died from prostate cancer and were sampled, provided a LOD score = +3.56 on chromosome arm 11p15. An 11p15 haplotype inherited in common from the founder to 6 generations of descendants is shared in at least 9 of the prostate cancer cases, 3 of whom died from prostate cancer. The 1-LOD drop defined support interval for the pedigree linked to chromosome 11 occurs at approximately 34-46 cM and contains 13 currently documented genomic features, several of which may be reasonable candidates for lethal prostate cancer including the gene PRMT3 (arginine methyltransferase 3), DAL-1, NELL1, FANCF (Fanconi anemia, complementation group F), and GAS2 (growth arrest-specific 2). Sequencing of multiple haplotype sharing cases in the pedigree is underway. Citation Format: Lisa A. Cannon-Albright, Craig C. Teerlink, Neeraj Agarwal. Identification of significant linkage evidence for lethal prostate cancer on chromosome arm 11p15. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1341. doi:10.1158/1538-7445.AM2013-1341

Protective Locus Against Renal Scarring on Chromosome 11 in Affected Sib Pairs with Familial Vesicoureteral Reflux Identified by Single Nucleotide Polymorphism Linkage Analysis

We identified the loci associated with renal scarring risk and protection in affected sib pairs with familial vesicoureteral reflux. A genome-wide analysis of vesicoureteral reflux with high density single nucleotide polymorphisms was conducted in 43 families with 2 or more affected children. A total of 43 probands and 58 affected siblings were included in the analysis. Genomic DNA was extracted from blood or saliva from all patients. All nuclear families had complete parental genotypes and all were Caucasian. Renal scarring was present in 23 of the 43 probands as detected by dimercapto-succinic acid imaging. easyLINKAGE software was used for the genome-wide linkage analysis. A LOD (logarithm [base 10] of odds) score of 3.3 or greater was considered significant evidence of linkage and a LOD score of 2.4 or greater but less than 3.3 was considered suggestive evidence of linkage. Using the affected sib pair method of analysis, a statistically significant linkage peak with a multipoint LOD score of 3.66 for patients without renal scarring was identified on chromosome 11 at 47.97 cM. For the scarring group a peak with a multipoint LOD score of 2.69 was identified on chromosome 17, which provides suggestive evidence of linkage. Our results suggest that a locus on chromosome 11 is associated with protection against renal scarring in patients with vesicoureteral reflux. In addition, a new locus on chromosome 17 may be linked to renal scarring. Our results suggest that multiple genes contribute to the formation of the vesicoureteral reflux phenotype, with patients having a unique susceptibility to renal injury/damage.

Genome-wide linkage and association scans for pulse pressure in Chinese twins

Elevated pulse pressure (PP) is associated with cardiovascular disorders and mortality in various populations. The genetic influence on PP has been confirmed by heritability estimates using related individuals. Recently, efforts have been made by mapping genes that are linked to the phenotype. We report the results of our gene mapping studies conducted in the Chinese population in mainland China. The genome-wide linkage and association scans were carried out on 63 middle-aged dizygotic twin pairs using high-density markers. The linkage analysis identified three significant linkage peaks (all with a single point P<1e(-05)) on chromosome 11 (LOD core 4.06 at 30.5 cM), chromosome 12 (LOD score 3.97 at 100.7 cM) and chromosome 18 (LOD score 4.01 at 70.7 cM), with the last two peaks closely overlapping with linkage peaks reported by two American studies. Multiple regions with suggestive linkages were identified, with many of the peaks overlapping with published linkage regions. The genome-wide association analysis detected a suggestive association on chromosome 4 (rs17031508, P<8.34e(-08)) located within a wide region of suggestive linkage. Our results provide some evidence for genetic linkages and associations with PP in the Chinese population. Further investigation is warranted to replicate the findings and to explore the susceptibility loci or genes for PP.

A Genome-Wide Analysis of Liberal and Conservative Political Attitudes

The assumption that the transmission of social behaviors and political preferences is purely cultural has been challenged repeatedly over the last 40 years by the combined evidence of large studies of adult twins and their relatives, adoption studies, and twins reared apart. Variance components and path modeling analyses using data from extended families quantified the overall genetic influence on political attitudes, but few studies have attempted to localize the parts of the genome which accounted for the heritability estimates found for political preferences. Here, we present the first genome-wide analysis of Conservative-Liberal attitudes from a sample of 13,000 respondents whose DNA was collected in conjunction with a 50-item sociopolitical attitude questionnaire. Several significant linkage peaks were identified and potential candidate genes discussed.