Peak LOD Score Research Articles

Characterization of chromosome 5q35 risk locus in African Ancestry population.

AbstractBackgroundAs part of the Research in African American Alzheimer Disease Initiative (REAAADI) and Late‐Onset AD Family Study (LOAD), genotyping array and whole genome sequencing (WGS) data were generated for 51 families (160 affected and 318 unaffected). Multipoint linkage analyses identified a peak LOD score on chr5q35 (HLOD=3.20). Additionally, a suggestive locus flanking the 1‐LOD score was previously identified in an AA GWAS study (p‐value=2.6×10‐6) (Kunkle et al, 2019). Here, we further characterize this locus and its role in AD.MethodWe performed region‐wide association analysis in an independent REAAADI dataset of ∼240 cases and ∼650 controls to fine map the signal in the locus. Additionally, we analyzed the WGS data to prioritize variants in the consensus regions based on segregation with disease among affected individuals and rarity (MAF<0.01). All variants were annotated for putative function including protein changes for coding variants and evidence for regulatory activity (ENCODE, RoadMap Epigenome) and chromatin interactions (publicly available HiC and promoter capture C) for noncoding variants.ResultRegion‐wide association analysis identified two regions of rare (MAF∼0.01) variants downstream (p=3.0×10‐8) and upstream (p=3.7×10‐6) of the 1‐LOD linkage region. Segregation analysis using WGS data identified 111 rare variants (MAF<0.01) segregating with disease in the AD individuals of the family with the highest LOD score contributing to the linkage peak. These include a 3’UTR and synonymous variant in INSYN2B/FAM196B as well as a promoter variant in WWC1/KIBRA. Interestingly, four other AA families contributing to the chr5 linkage signal harbor different within‐family shared variants located in INSYN2B’s promoter or in enhancer regions with evidence for interaction with INSYN2B’s promoter. WWC1, expressed in astrocytes, was previously reported in AD context, whereas novel candidate INSYN2B encodes an inhibitory synaptic factor active in oligodendrocytes and neurons.ConclusionOur initial analyses provide evidence for two candidate genes contributing to AD genetics in the AA population. Additional work for functional validation of these candidates for AD is ongoing. This AA population‐specific finding shows the importance of diversifying population‐level genetic data to better understand the genetic determinants of AD on a global scale.

Pycard and BC017158 Candidate Genes of Irm1 Locus Modulate Inflammasome Activation for IL-1β Production.

We identified Pycard and BC017158 genes as putative effectors of the Quantitative Trait locus (QTL) that we mapped at distal chromosome 7 named Irm1 for Inflammatory response modulator 1, controlling acute inflammatory response (AIR) and the production of IL-1β, dependent on the activation of the NLRP3 inflammasome. We obtained the mapping through genome-wide linkage analysis of Single Nucleotide Polymorphisms (SNPs) in a cross between High (AIRmax) and Low (AIRmin) responder mouse lines that we produced by several generations of bidirectional selection for Acute Inflammatory Response. A highly significant linkage signal (LOD score peak of 72) for ex vivo IL-1β production limited a 4 Mbp interval to chromosome 7. Sequencing of the locus region revealed 14 SNPs between “High” and “Low” responders that narrowed the locus to a 420 Kb interval. Variants were detected in non-coding regions of Itgam, Rgs10 and BC017158 genes and at the first exon of Pycard gene, resulting in an E19K substitution in the protein ASC (apoptosis associated speck-like protein containing a CARD) an adaptor molecule in the inflammasome complex. Silencing of BC017158 inhibited IL1-β production by stimulated macrophages and the E19K ASC mutation carried by AIRmin mice impaired the ex vivo IL-1β response and the formation of ASC specks in stimulated cells. IL-1β and ASC specks play major roles in inflammatory reactions and in inflammation-related diseases. Our results delineate a novel genetic factor and a molecular mechanism affecting the acute inflammatory response.

Abstract 2920: A Diversity outbred (DO) mouse model reveals regulators of HER2-driven tumorigenesis

Abstract Although humans are heterogeneous, evaluation of tumorigenesis and immunotherapy has relied on the use of inbred mouse strains, which may lead to biased results. In order to investigate the effect(s) of genetic background on tumor incidence and immune response, we have taken a unique new approach to identify genes that are associated with tumor onset time & tumor growth rate using Diversity Outbred (DO) mice. Specifically, we crossed DO female mice (J:DO, Jackson Labs) with BALB/NeuT (NeuT) mice to introduce a heterogeneous background in (BALBxDO) F1 NeuT mice. NeuT express transforming rat neu, the homologue of HER2, and develop spontaneous tumors in all 10 mammary glands between 14 and 19 weeks of age (woa). DO mice are generated by non-sibling crosses of 8 founder strains: A/J, C57BL/6J, 129S1/SvlmJ, NOD/ShiLtJ, NZO/HILtJ, CAST/EiJ, PWK/PhJ, WSB/EiJ to encompass greater than 90% of murine polymorphic alleles. The genetic makeup of each F1 mouse is defined using 143,600 chromosomal markers (mostly SNP) in the Giga Mouse Universal Genotyping Array. SNP analysis identifies the origin of quantitative trait loci (QTL), using the R/QTL package. The program reads the GigaMUGA genotyping results to perform haplotype reconstruction and determine the founder strain haplotype of each marker locus. DO F1 mice have been successfully used to identify the critical role of MHC-IB, and NK cells, in generation of HER2 Ab response following vaccination of HER2 tolerant mice (Wei et al., 2020). In (BALBxDO) F1 NeuT female mice most spontaneous mammary tumors develop over a wider range of time from 9 to 23 woa. QTL analysis of tumor onset time revealed QTLs in chromosomes (Chr) 1 and X with peak LOD scores of 7.7 (p = 0.01) and 8.6 (p < 0.05), respectively. Tumor growth rate was associated with a QTL in Chr 10. More specifically, PWK and CAST haplotypes in these QTLs were associated with early and faster growing tumors. About 10-15 genes in the 3 QTLs contain missense SNP alterations unique to PWK and CAST, and the genes have been associated with human breast cancer prognosis. As an example, Mid2 identified in ChrX QTL interacts with BRCA1, and is negatively associated with patient survival using the TCGA BRCA database. Single cell RNA sequencing revealed that Mid2 is expressed in NeuT mammary tumor tissue. Studies are ongoing to determine the functional significance of candidate genes in HER2/neu mammary tumorigenesis. DO F1 QTL combined with scRNA transcriptomic analysis opens important new opportunities to define critical genes in cancer biology and immunotherapy, with the potential for novel intervention and disease prevention. Citation Format: Jennifer Jacob, Kuang Wei, Joyce D. Reyes, Cong-Cong Yin, Elizabeth Rondini, Qing-Sheng Mi, James Granneman, Prahlad Parajuli, K Wagner, Wei-Zen Wei. A Diversity outbred (DO) mouse model reveals regulators of HER2-driven tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2920.

Novel caries loci in children and adults implicated by genome-wide analysis of families

BackgroundDental caries is a common chronic disease among children and adults alike, posing a substantial health burden. Caries is affected by multiple genetic and environmental factors, and prior studies have found that a substantial proportion of caries susceptibility is genetically inherited.MethodsTo identify such genetic factors, we conducted a genome-wide linkage scan in 464 extended families with 2616 individuals from Iowa, Pennsylvania and West Virginia for three dental caries phenotypes: (1) PRIM: dichotomized as zero versus one or more affected primary teeth, (2) QTOT1: age-adjusted quantitative caries measure for both primary and permanent dentitions including pre-cavitated lesions, and (3) QTOT2: age-adjusted quantitative caries excluding pre-cavitated lesions. Genotyping was conducted for approximately 600,000 SNPs on an Illumina platform, pruned to 127,511 uncorrelated SNPs for the analyses reported here.ResultsMultipoint non-parametric linkage analyses generated peak LOD scores exceeding 2.0 for eight genomic regions, but no LOD scores above 3.0 were observed. The maximum LOD score for each of the three traits was 2.90 at 1q25.3 for PRIM, 2.38 at 6q25.3 for QTOT1, and 2.76 at 5q23.3 for QTOT2. Some overlap in linkage regions was observed among the phenotypes. Genes with a potential role in dental caries in the eight chromosomal regions include CACNA1E, LAMC2, ALMS1, STAMBP, GXYLT2, SLC12A2, MEGF10, TMEM181, ARID1B, and, as well as genes in several immune gene families. Our results are also concordant with previous findings from association analyses on chromosomes 11 and 19.ConclusionsThese multipoint linkage results provide evidence in favor of novel chromosomal regions, while also supporting earlier association findings for these data. Understanding the genetic etiology of dental caries will allow designing personalized treatment plans based on an individual’s genetic risk of disease.

A Locus on Chromosome 5 Is Associated with Red Cell Allo-Immunization in Adults with Sickle Cell Disease

Background: Transfusion is a standard treatment for acute and chronic complications of sickle cell disease (SCD). This therapy is limited by a high prevalence of red cell antigen alloimmunization, which is not seen in other frequently transfused patient populations. Reasons for this increased prevalence have not been elucidated. One model based study suggested a genetic predisposition in all patient populations. Other studies have provided evidence that antigen mismatches occur commonly between blood donors of European ancestry and SCD patients and that this is a primary cause for alloimmunization. Accordingly, limited phenotypic antigen matching is a standard practice used to minimize alloimmunization risk. We hypothesized that if these population differences are the biologic basis for this phenomenon that admixture mapping could identify significant susceptibility loci.Methods: A cohort study consisting of adults evaluated at NIH and Howard University between 2001-2015 (n=721) had allo-antibody status confirmed with the blood bank at the hospital where the most recent transfusion was performed. Genomic DNA was genotyped for a panel of 1622 ancestry informative SNPs in 359 patients. Genotypes were used to infer ancestry proportions using Structure software assuming a 3 population model. Ancestry was analyzed by regression along with clinical variables. Genome wide admixture mapping was performed to identify regions of the genome where ancestry differences between cases and controls suggested an association. We used AncestryMap for a log-additive case control study assuming various risk models.Results: Five hundred fifty four subjects (77% of cohort) had a transfusion history with 153 (27.6 %) with allo-antibodies and 65 (11.7 %) with no prior transfusions. Antibodies directed against RH, Kell, MNS and Duffy antigens were the most prevalent. Clinical factors associated with alloimmunization in regression models were only significant for lifetime transfusion history by both logistic (antibody status, β=0.56, P=6.31 x 10-6) and linear (number of antibodies, β=0.35, P=0.0004) regressions. When considering genotype data, Amerindian ancestry was associated with antibody status (logistic, β=4.60, P=0.03) and number of antibodies (linear, β=4.92, P=0.006), while African and European ancestry were not. The association between ancestry and alloimmunization risk remained unchanged when the number of prior transfusions was also included in both multivariable models for alloimmunization status and number of antibodies, P=0.09 and P=0.06, respectively. Genome wide mapping by admixture linkage disequilibrium compared 91 allo-Ab cases to 210 controls with prior transfusions under 10 different risk models (range 0.25-9.0). The most significant result was the 0.25 risk model which was highly suggestive for association (genome log factor 10.96, P<0.001; Figure 1A). This scan identified region of chromosome 5 (5q21.3-5q31.1) defined by 14 snps spanning 21.685 Mb and a highly significant peak LOD score of 13.5 (Figure 1B). While this region contains >540 genes, the linkage region is noteworthy for a cytokine gene cluster implicated in hematopoiesis and immune responses.Conclusions: In a large cohort of adults with SCD, the prevalence of red cell alloimmunization is 27.6%. Amerindian ancestry is a risk factor and suggests a genetic component to this phenotype. The number of prior transfusions is also a significant risk factor. Genome wide admixture mapping identified a strong association at chromosome 5q21.3-5q31.1 using a 0.25 risk model. This chromosome 5 association is particularly interesting given the presence a cytokine gene cluster within a portion of the associated region. Analysis of larger, admixed populations will help to fine map allo-immunization susceptibility variants within specific genes that underlie these loci. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Reduced brain UCP2 expression mediated by microRNA-503 contributes to increased stroke susceptibility in the high-salt fed stroke-prone spontaneously hypertensive rat

UCP2 maps nearby the lod score peak of STR1-stroke QTL in the SHRSP rat strain. We explored the potential contribution of UCP2 to the high-salt diet (JD)-dependent increased stroke susceptibility of SHRSP. Male SHRSP, SHRSR, two reciprocal SHRSR/SHRSP-STR1/QTL stroke congenic lines received JD for 4 weeks to detect brain UCP2 gene/protein modulation as compared with regular diet (RD). Brains were also analyzed for NF-κB protein expression, oxidative stress level and UCP2-targeted microRNAs expression level. Next, based on knowledge that fenofibrate and Brassica Oleracea (BO) stimulate UCP2 expression through PPARα activation, we monitored stroke occurrence in SHRSP receiving JD plus fenofibrate versus vehicle, JD plus BO juice versus BO juice plus PPARα inhibitor. Brain UCP2 expression was markedly reduced by JD in SHRSP and in the (SHRsr.SHRsp-(D1Rat134-Mt1pa)) congenic line, whereas NF-κB expression and oxidative stress level increased. The opposite phenomenon was observed in the SHRSR and in the (SHRsp.SHRsr-(D1Rat134-Mt1pa)) reciprocal congenic line. Interestingly, the UCP2-targeted rno-microRNA-503 was significantly upregulated in SHRSP and decreased in SHRSR upon JD, with consistent changes in the two reciprocal congenic lines. Both fenofibrate and BO significantly decreased brain microRNA-503 level, upregulated UCP2 expression and protected SHRSP from stroke occurrence. In vitro overexpression of microRNA-503 in endothelial cells suppressed UCP2 expression and led to a significant increase of cell mortality with decreased cell viability. Brain UCP2 downregulation is a determinant of increased stroke predisposition in high-salt-fed SHRSP. In this context, UCP2 can be modulated by both pharmacological and nutraceutical agents. The microRNA-503 significantly contributes to mediate brain UCP2 downregulation in JD-fed SHRSP.

Distinct Genetic Loci Are Associated with Platelet Aggregation Responses to Collagen and Collagen Synthetic Related Peptide (SRP) in Mice

There is substantial inter-individual variation in responsiveness of human platelets to different agonists in platelet aggregation studies. Much of this variation is likely heritable, and some of this variation can be attributed to genetic variants in platelet agonist receptors. However, beyond variants in known candidate receptor genes, the genetic factors underlying variation in platelet aggregation are undefined. In this study, we are taking a genome wide approach to identify genetic factors associated with platelet aggregation responses to different agonists in mice. We developed a method to quantify platelet aggregation responses to multiple agonists in single mouse donors and tested platelets from C57BL6/J (B6) and FVB/NJ (FVB) inbred mouse strains for their patterns of response to collagen (type I), collagen synthetic reactive peptide (SRP), the thromboxane A2 (TxA2) mimetic U44069, and PAR-4 activating peptide (AYPGKF) (B6 and FVB, n=9 for each strain). The platelet aggregation response was then quantified as an area under the aggregation curve (AUC) for each agonist. There was a significant difference between B6 and FVB in platelet responsiveness to collagen (9.06 x 103 ± 1.44 x 103 vs 3.7 x 103 ± 1.77 x 103. p<0.018), collagen SRP (2.04 x 103 ± 1.31 x 103 vs 1.25 x 104 ± 1.55 x 103. p<5 x 10-5), and U44069 (1.2 x 104 ± 2.19 x 103 vs 6.47 x 103 ± 1.85 x 103. p<0.05), respectively. No difference was observed in response to PAR-4 AP (7.07 x 103 ± 6.92 x 102 vs 8.24 x 103 ± 1.25 x 103. p=0.2). We then performed a B6 x FVB F2 intercross which generated 152 mice which were phenotyped for platelet aggregation responsiveness to collagen, collagen SRP, and U44069, using AYPGKF as a positive control. Thus far, we have performed genome-wide association studies in 54 mice using a 1449 SNP Illumina BeadChip platform. Quantitative trait loci (QTL) analysis was performed to identify QTLs associated with platelet responsiveness to collagen, SRP, and U44069. In this interim analysis, we have identified several significant QTL for platelet aggregation response to collagen and SRP. A single highly significant QTL for collagen SRP was identified on proximal chromosome 7 (LOD = 8.26) that spans ~23 Mb (5’ end to rs6239372), termed Plsrp1. Additionally, statistically significant but broad QTLs were identified for collagen on chromosome 14 (Plcoll1 and Plcoll2, peak LOD scores 3.78 and 3.87, respectively), with additional suggestive regions on chromosome 10 and chromosome 7. Intriguingly, the QTL with the strongest associations for collagen on chromosome 14 (Plcoll1 and Plcoll2) are necessarily distinct from the single collagen SRP QTL (Plsrp1) on chromosome 7. Interestingly, Plcoll1 and Plcoll2 would have been suspected from previous studies of collagen responsiveness in mice as chromosome 14 does not harbor the major candidate genes thought to interact with collagen, in contrast to Plsrp1 which contains Gp6. These findings indicate distinct and previously unsuspected genetic elements underlie the differences in platelet responsiveness to collagen and collagen SRP in mice. DisclosuresNo relevant conflicts of interest to declare.

Genetic control of renal tumorigenesis by the mouse Rtm1 locus

BackgroundThe genetic basis of susceptibility to renal tumorigenesis has not yet been established in mouse strains. Mouse lines derived by bidirectional phenotypic selection on the basis of their maximal (AIRmax) or minimal (AIRmin) acute inflammatory responsiveness differ widely in susceptibility to spontaneous and urethane-induced renal tumorigenesis. To map the functional loci modulating renal tumor susceptibility in these mice, we carried out a genome-wide genetic linkage study, using SNP arrays, in an (AIRmax x AIRmin)F2 intercross population treated with a single urethane dose at 1 week of age and phenotyped for renal tumors at 35 weeks of age.ResultsAIRmax mice did not develop renal tumors spontaneously nor in response to urethane, whereas in AIRmin mice renal tumors formed spontaneously (in 52% of animals) and after urethane induction (89%). The tumors had a papillary morphology and were positive for alpha-methylacyl-CoA racemase and negative for CD10. By analysis of 879 informative SNPs in 662 mice, we mapped a single quantitative trait locus modulating the incidence of renal tumors in the (AIRmax x AIRmin)F2 intercross population. This locus, which we named Renal tumor modifier QTL 1 (Rtm1), mapped to chromosome 17 at 23.4 Mb (LOD score = 15.8), with SNPs rs3696835 and rs3719497 flanking the LOD score peak. The A allele of rs3719497 from AIRmin mice was associated with a 2.5-fold increased odds ratio for renal tumor development. The LOD score peak included the Tuberous sclerosis 2 (Tsc2) gene which has already been implicated in kidney disease: loss of function by germline retroviral insertion is associated with spontaneous renal tumorigenesis in the Eker rat, and heterozygous-null Tsc2(+/-) mice develop renal cystadenomas.ConclusionsWe mapped Rtm1 as a single major locus modulating renal tumorigenesis in a murine intercross population. Thus, the AIR mouse lines can be considered a new genetic model for studying the role of germline and somatic molecular alterations in kidney neoplastic disease.

Obese Locus in WNIN/Obese Rat Maps on Chromosome 5 Upstream of Leptin Receptor

WNIN/Obese (WNIN/Ob) rat a new mutant model of metabolic syndrome was identified in 1996 from an inbred Wistar rat strain, WNIN. So far several papers are published on this model highlighting its physical, biochemical and metabolic traits. WNIN/Ob is leptin resistant with unaltered leptin or its receptor coding sequences - the two well-known candidate genes for obesity. Genotyping analysis of F2 progeny (raised from WNIN/Ob × Fisher - 344) in the present study localized the mutation to a recombinant region of 14.15cM on chromosome 5. This was further corroborated by QTL analysis for body weight, which narrowed this region to 4.43 cM with flanking markers D5Rat256 & D5Wox37. Interval mapping of body weight QTL shows that the LOD score peak maps upstream of leptin receptor and shows an additive effect suggesting this as a novel mutation and signifying the model as a valuable resource for studies on obesity and metabolic syndrome.

Nonsyndromic brachydactyly type D and type E mapped to 7p15 in healthy children and adults from the Jirel ethnic group in eastern Nepal.

There is phenotypic overlap between Brachydactyly Type D (BDD) and Brachydactyly Type E (BDE) that suggests a possible common underlying etiology. We seek to understand the genetic underpinnings of, and relationship between, these skeletal anomalies. The Jirel ethnic group of eastern Nepal participates in various genetic epidemiologic studies, including those in which hand-wrist radiographs have been taken to examine skeletal development. Nearly 2,130 individuals (969 males; 1,161 females) were phenotyped for BDD/BDE. Of these, 1,722 individuals (773 males; 949 females) were genotyped for 371 STR markers spanning the autosomal genome. Variance components-based linkage analysis was used to conduct a genome-wide linkage scan for QTL influencing the BDD/BDE phenotype. BDD was present in 3.55%, and BDE was present in 0.39%, of the study sample. Because of the phenotypic overlap between two traits, affecteds of either type were considered as affected by a single combined phenotype (BDD/BDE) having a prevalence of 3.94%. The additive genetic heritability of BDD/BDE was highly significant (h(2) ± SE = 0.89 ± 0.13; P = 1.7 × 10(-11) ). Significant linkage of BDD/BDE was found to markers on chromosome 7p21-7p14 (peak LOD score = 3.74 at 7p15 between markers D7S493 and D7S516). Possible positional candidate genes in the one-lod support interval of this QTL include TWIST and the HOXA1-A13 cluster. This is the first study to report significant linkage results for BDD/BDE using a large extended pedigree, and the first to suggest that mutations in TWIST and/or the HOXA1-A13 cluster may contribute to these specific skeletal anomalies.

A new locus for familial temporal lobe epilepsy on chromosome 3q

Temporal lobe epilepsy (TLE) is a common and heterogeneous focal epilepsy syndrome with a complex etiology, involving both environmental and genetic factors. Several familial forms of TLE have been described, including familial lateral TLE (FLTLE), familial mesial TLE (FMTLE) without hippocampal sclerosis, and FMTLE with hippocampal sclerosis. Mutations have been identified only in the leucine-rich, glioma-inactivated 1 (LGI1) gene on chromosome 10q22-q24 in FLTLE. Several loci have been mapped in families with FMTLE, but responsible genes have not been found. We report clinical evaluation in a large family with FMTLE and a new genetic locus. We conducted a genome-wide scan using 10cM-spaced microsatellite markers on a family with TLE. Seven individuals had TLE without antecedent FS; four other individuals had FS during childhood, but no subsequent epilepsy. Patients with TLE had infrequent simple partial, complex partial and secondarily generalized seizures that generally responded well to treatment. The proband had no hippocampal sclerosis. The mode of inheritance appeared to be autosomal dominant with incomplete penetrance. Linkage analysis was performed using the Genehunter software. Regions with LOD score>1 and those that were poorly informative in the first-pass scan were further genotyped. Linkage was identified on chromosome 3q25-q26 in a 13cM region flanked by markers D3S1584 and D3S3520, with a peak LOD score of 3.23. This interval does not correspond to any previously known locus for familial epilepsy or FS. KCNAB1, encoding a voltage-gated, shaker-related potassium channel, and NLGN1, encoding a member of a family of neuronal cell surface protein were excluded as disease causing mutations. We identified a novel locus for familial TLE with FS, providing additional evidence of the complexity and genetic heterogeneity of familial focal epilepsy.

Mutations in the autoregulatory domain of β-tubulin 4a cause hereditary dystonia.

Dystonia type 4 (DYT4) was first described in a large family from Heacham in Norfolk with an autosomal dominantly inherited whispering dysphonia, generalized dystonia, and a characteristic hobby horse ataxic gait. We carried out a genetic linkage analysis in the extended DYT4 family that spanned 7 generations from England and Australia, revealing a single LOD score peak of 6.33 on chromosome 19p13.12-13. Exome sequencing in 2 cousins identified a single cosegregating mutation (p.R2G) in the β-tubulin 4a (TUBB4a) gene that was absent in a large number of controls. The mutation is highly conserved in the β-tubulin autoregulatory MREI (methionine–arginine–glutamic acid–isoleucine) domain, highly expressed in the central nervous system, and extensive in vitro work has previously demonstrated that substitutions at residue 2, specifically R2G, disrupt the autoregulatory capability of the wild-type β-tubulin peptide, affirming the role of the cytoskeleton in dystonia pathogenesis.

Systems Genetics of Metabolism: The Use of the BXD Murine Reference Panel for Multiscalar Integration of Traits

Metabolic homeostasis is achieved by complex molecular and cellular networks that differ significantly among individuals and are difficult to model with genetically engineered lines of mice optimized to study single gene function. Here, we systematically acquired metabolic phenotypes by using the EUMODIC EMPReSS protocols across a large panel of isogenic but diverse strains of mice (BXD type) to study the genetic control of metabolism. We generated and analyzed 140 classical phenotypes and deposited these in an open-access web service for systems genetics (www.genenetwork.org). Heritability, influence of sex, and genetic modifiers of traits were examined singly and jointly by using quantitative-trait locus (QTL) and expression QTL-mapping methods. Traits and networks were linked to loci encompassing both known variants and novel candidate genes, including alkaline phosphatase (ALPL), here linked to hypophosphatasia. The assembled and curated phenotypes provide key resources and exemplars that can be used to dissect complex metabolic traits and disorders.

Putative resistance gene markers associated with quantitative trait loci for fire blight resistance in Malus 'Robusta 5' accessions.

BackgroundBreeding of fire blight resistant scions and rootstocks is a goal of several international apple breeding programs, as options are limited for management of this destructive disease caused by the bacterial pathogen Erwinia amylovora. A broad, large-effect quantitative trait locus (QTL) for fire blight resistance has been reported on linkage group 3 of Malus ‘Robusta 5’. In this study we identified markers derived from putative fire blight resistance genes associated with the QTL by integrating further genetic mapping studies with bioinformatics analysis of transcript profiling data and genome sequence databases.ResultsWhen several defined E.amylovora strains were used to inoculate three progenies from international breeding programs, all with ‘Robusta 5’ as a common parent, two distinct QTLs were detected on linkage group 3, where only one had previously been mapped. In the New Zealand ‘Malling 9’ X ‘Robusta 5’ population inoculated with E. amylovora ICMP11176, the proximal QTL co-located with SNP markers derived from a leucine-rich repeat, receptor-like protein ( MxdRLP1) and a closely linked class 3 peroxidase gene. While the QTL detected in the German ‘Idared’ X ‘Robusta 5’ population inoculated with E. amylovora strains Ea222_JKI or ICMP11176 was approximately 6 cM distal to this, directly below a SNP marker derived from a heat shock 90 family protein gene ( HSP90). In the US ‘Otawa3’ X ‘Robusta5’ population inoculated with E. amylovora strains Ea273 or E2002a, the position of the LOD score peak on linkage group 3 was dependent upon the pathogen strains used for inoculation. One of the five MxdRLP1 alleles identified in fire blight resistant and susceptible cultivars was genetically associated with resistance and used to develop a high resolution melting PCR marker. A resistance QTL detected on linkage group 7 of the US population co-located with another HSP90 gene-family member and a WRKY transcription factor previously associated with fire blight resistance. However, this QTL was not observed in the New Zealand or German populations.ConclusionsThe results suggest that the upper region of ‘Robusta 5’ linkage group 3 contains multiple genes contributing to fire blight resistance and that their contributions to resistance can vary depending upon pathogen virulence and other factors. Mapping markers derived from putative fire blight resistance genes has proved a useful aid in defining these QTLs and developing markers for marker-assisted breeding of fire blight resistance.

Fine mapping of dental fluorosis quantitative trait loci in mice

Genetic factors underlie the susceptibility and the resistance to dental fluorosis (DF). The A/J (DF susceptible) and 129P3/J (DF resistant) mouse strains have previously been used to detect quantitative trait loci (QTLs) associated with DF on chromosome (Chr) 2 and Chr 11. In the present study, increased marker density genotyping followed by interval mapping was performed to narrow the QTL intervals and improve the logarithm of the odds (to the base 10) (LOD) scores. Narrower intervals were obtained on Chr 2 where LOD ≥ 6.0 (57-84 cM or ≈ 51 Mb), LOD ≥ 7.0 (62-79 cM or ≈ 32 Mb), and LOD ≥ 8.0 (65-74 cM or ≈ 17 Mb); and on Chr 11 where LOD ≥ 6.0 (18-51 cM or ≈ 53 Mb), LOD ≥ 7.0 (28-48 cM or ≈ 34 Mb), and LOD ≥ 8.0 (31-45 cM or ≈ 22 Mb). Haplotype analysis between A/J and 129P3/J mice further reduced the QTL intervals. Accn1 was selected as a candidate gene based upon its location near the peak LOD score on Chr 11 and distant homology with the Caenorhabditis elegans fluoride-resistance gene, flr1. The severity of DF between Accn1(-/-) and wild-type mice was not significantly different. Hence, the loss of ACCN1 function does not modify DF severity in mice. Narrowing the DF QTL intervals will facilitate additional candidate gene selections and interrogation.

A QTL for rice grain yield in aerobic environments with large effects in three genetic backgrounds

A large-effect QTL associated with grain yield in aerobic environments was identified in three genetic backgrounds, Apo/(2)*Swarna, Apo/(2)*IR72, and Vandana/(2)*IR72, using bulk-segregant analysis (BSA). Apo and Vandana are drought-tolerant aerobic-adapted varieties, while Swarna and IR72 are important lowland rice varieties grown on millions of hectares in Asia but perform poorly in aerobic conditions. Two closely linked rice microsatellite (RM) markers, RM510 and RM19367, located on chromosome 6, were found to be associated with yield under aerobic soil conditions in all three backgrounds. The QTL linked to this marker, qDTY6.1 (DTY, grain yield under drought), was mapped to a 2.2cM region between RM19367 and RM3805 at a peak LOD score of 32 in the Apo/(2)*Swarna population. The effect of qDTY6.1 was tested in a total of 20 hydrological environments over a period of five seasons and in five populations in the three genetic backgrounds. In the Apo/(2)*Swarna population, qDTY6.1 had a large effect on grain yield under favorable aerobic (R (2)≤66%) and irrigated lowland (R (2)<39%) conditions but not under drought stress; Apo contributed the favorable allele in all the conditions where an effect was observed. In the Apo/IR72 cross, Apo contributed the favorable allele in almost all the aerobic environments in RIL and BC(1)-derived populations. In the Vandana/IR72 RIL and BC(1)-derived populations, qDTY6.1 had a strong effect on yield in aerobic drought stress, aerobic non-stress, and irrigated lowland conditions; the Vandana allele was favorable in aerobic environments and the IR72 allele was favorable in irrigated lowland environments. We conclude that qDTY6.1 is a large-effect QTL for rice grain yield under aerobic environments and could potentially be used in molecular breeding of rice for aerobic environments.

Identification of the nephropathy-susceptibility locus HIVAN4.

HIVAN1, HIVAN2, and HIVAN3 are nephropathy-susceptibility loci previously identified in the HIV-1 transgenic mouse, a model of collapsing glomerulopathy. The HIVAN1 and HIVAN2 loci modulate expression of Nphs2, which encodes podocin and several other podocyte-expressed genes. To identify additional loci predisposing to nephropathy, we performed a genome-wide scan in 165 backcross mice generated between the nephropathy-sensitive HIV-1-transgenic FVB/NJ (TgFVB) strain and the resistant Balb/cJ (BALB) strain. We identified a major susceptibility locus (HIVAN4) on chromosome 6 G3-F3, with BALB alleles conferring a twofold reduction in severity (peak LOD score = 4.0). Similar to HIVAN1 and HIVAN2, HIVAN4 modulated expression of Nphs2, indicating a common pathway underlying these loci. We independently confirmed the HIVAN4 locus in a sister TgFVB colony that experienced a dramatic loss of nephropathy subsequent to a breeding bottleneck. In this low-penetrance line, 3% of the genome was admixed with BALB alleles, suggesting a remote contamination event. The admixture localized to discrete segments on chromosome 2 and at the HIVAN4 locus. HIVAN4 candidate genes include killer lectin-like receptor genes as well as A2m and Ptpro, whose gene products are enriched in the glomerulus and interact with HIV-1 proteins. In summary, these data identify HIVAN4 as a major quantitative trait locus for nephropathy and a transregulator of Nphs2. Furthermore, similar selective breeding strategies may help identify further susceptibility loci.

E Integrative genomics approaches identify new genes controlling heart rate

IntroductionHeart rate (HR) is a fundamental measure of cardiac function, and is of prognostic and therapeutic significance. We applied genetic and genomic approaches to identify new loci and genes controlling...

Genetic Investigation by Shared Genomic Segment and Linkage Study of a Unique Family with Primary Familial and Congenital Polycythemia

AbstractAbstract 4783 Introduction:Polycythemia/Erythrocytosis may be congenital and/or familial, or acquired. The first recognized familial polycythemias are associated with mutations in the globins gene resulting in increased affinity of Hb to oxygen, which could also be caused by congenital deficiency of 2, 3 DPG. Subsequently gain-of-function of erythropoietin receptor gene mutations have been shown to cause familial dominantly inherited polycythemia. More recently, mutation in the genes involved in hypoxia sensing pathway have been described as causes of congenital polycythemia; these include recessively inherited VHL (Chuvash polycythemia), and dominantly inherited HIF2a and PHD2 mutations. Yet unexplained familial polycythemias still exist. Here we investigated a family with dominant polycythemia present in five generations wherein all known mutations in polycythemia-causing genes were excluded through linkage analyses and direct sequencing. Familial clustering of the disease provided us an opportunity to identify a novel genetic cause modulating erythropoiesis. We utilized a shared genomic segment (SGS) and linkage analysis approach, to provide evidence of chromosomal candidate regions for further efforts to localize the causative gene. Methods:A five generation family pedigree consisting of 26 phenotyped members (10 affected/16 unaffected) with 16 subjects (5 affected and 11 unaffected) genotyped on an Affymetrix v5.0 SNP platform. A conventional two-point linkage analysis did not identify a potential causative genomic region. Multipoint linkage analysis was subsequently performed because this allows for optimal use of the genetic SNP data to infer inheritance patterns and leads to lower false positive rates than two-point linkage methods. In order to identify potential causative regions for harboring the polycythemia-causing defect, we also performed shared genomic segment (SGS) analysis that recognizes a region identical by descent must also be shared by identical by state. We analyzed 409,426 reference dbSNPs with a 94% call rate for genotyped subjects to determine runs of loci at which affected individuals share a common allele. Parametric multipoint linkage analysis used a pruned set of 8,394 non-monomorphic markers with minimum spacing of 100 kb, minimum heterozygosity of 0.3 and maximum pairwise r2 of 0.16 based on HapMap CEU data. We used a Markov chain Monte Carlo linkage method (MCLINK) which allows for fully informative multilocus linkage analysis on extended pedigrees. Results:SGS identified five runs of shared loci that achieved chromosome-wide significance among all five affected subjects (chromosome, run length): 18, 1980 SNPs; 14, 1292 SNPs and 1095 SNPs; 16, 1060 SNPs and 20, 500 SNPs. The SGS regions on chr. 14 (1292 and 1095 run lengths out of 13,149 total SNPs) begins at (NCBI build 36.1) 55,956,582,765bp (rs17254216) through 73,408,302bp (rs1778222) and contain regions of 80 and 79 consecutive SNPs at which all cases share homozygously from a homozygous shared genomic segment analysis (HGS). Multipoint linkage identified a peak LOD score of 3.41 on chr. 16 at 78,033,240bp (rs4888031) achieving genomewide significance which is contained within an observed SGS region of interest (77,507,467bp to 81,049,285bp). [Display omitted] Conclusion:We submit that genetic epidemiological analyses (SGS and Linkage) augment likelihood of identifying the disease-causing genomic regions that are good candidates for the underlying familial susceptibility variant(s). We observed an 8Mb region of interest on chromosome 14 which may contain a segment of loss-of-heterozygosity, and a 3.9Mb region on chromosome 16 both are now being analyzed by sequencing studies. Disclosures:No relevant conflicts of interest to declare.

Genome-Wide Linkage Analysis Reveals Novel Loci Modifying Plasma Von Willebrand Factor Undetected by Genome-Wide Association

AbstractAbstract 2116Plasma levels of von Willebrand Factor (VWF) are highly variable among healthy people and ~70% of this variability is heritable. Elevated levels of VWF are associated with thrombosis while low levels of VWF cause the common bleeding disorder, von Willebrand Disease (VWD). The genome-wide association study (GWAS) is a powerful tool for identifying common genetic variants contributing to complex traits, though less common variants are likely to be missed by this approach. We performed combined GWAS and linkage analysis in a cohort of healthy young college students. The study was approved by the University of Michigan IRB (IRBMED #2005-0080). 1189 healthy individuals (14-35 yrs of age) were recruited, comprising 507 sibships. VWF levels were determined by alphalisa assay (mean 109%, range 26 – 410%). VWF level heritability (h2) was 73% among siblings and 82% among monozygotic twins. Samples were de-identified and DNA was extracted from peripheral blood, quantified and genotyped using the Illumina Omni-1 quad SNP chip at the Broad Institute (MIT, Boston). 1182 individuals passed genotyping quality control with >97% call rates for 979,855 SNPs. Genotyping quality control was performed as part of the NIH's GENEVA program. GWAS on log transformed, age and sex adjusted VWF levels identified a major peak over the ABO locus (p<1×10-36), [Figure 1] confirming the known influence of ABO blood type on plasma VWF. Preliminary genome-wide linkage analysis (MERLIN) performed on plasma VWF levels in the 507 sibships identified 5 novel loci with peak LOD scores greater than 5 (chromosomes 2, 7, and 21), in addition to the ABO locus on chromosome 9 [Figure 2]. Other than ABO, none of these loci were detected in our GWAS analysis, or the previously reported CHARGE Consortium VWF GWAS (Smith, N et al. Circulation, 2010). Our analysis is now being extended to additional hemostasis and other related phenotypes using the same SNP data. In conclusion, linkage analysis identified novel loci contributing to the variation of VWF in healthy siblings that were undetected by GWAS. Though GWAS is an effective tool for the analysis of many complex traits, including plasma VWF variation, the majority of the heritable component of these traits remains unexplained. However, the missing heritability may be due, at least in part, to multiple rare variants at a limited number of loci. Our results suggest that genome-wide linkage analysis in multiple sibships may identify such loci and that this approach may be broadly applicable to hemostasis and other complex traits. Identification of the major loci regulating plasma VWF levels may lead to an improved prediction of bleeding and thrombotic risk and could suggest novel targets for therapeutic intervention. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.