Identification of genetic loci affecting mouse-adapted bovine spongiform encephalopathy incubation time in mice

Prion diseases are fatal neurodegenerative disorders of humans and animals, which include bovine spongiform encephalopathy (BSE) and its human form, variant Creutzfeldt-Jakob disease (vCJD). They are characterized by a prolonged incubation period, which is known to be influenced by polymorphisms in the prion protein gene. Previous studies of inbred mice have demonstrated that additional genetic loci also contribute to the observed variation in incubation period. However, a substantial transmission barrier between cow and mouse complicates studies using BSE. As a result, primary transmissions display large variations in incubation period and not all animals develop clinical signs of disease. To identify quantitative trait loci for BSE without the presence of a transmission barrier, we analysed 124 animals from an F2 intercross between CAST/Ei and NZW/OlaHsd mice and challenged them intracerebrally with a strain of BSE that was passaged twice through C57BL/6OlaHsd mice. Interval mapping identified two highly significant linked regions on chromosomes 2 and 11 with peak lod scores of 6.34 and 4.77, respectively. Composite interval mapping suggests that chromosome 2 includes three linked quantitative trait loci. Loci in the same position on chromosomes 2 and 11 were also identified in a previous study using the same mouse cross but infected with Chandler/RML scrapie prions. If these are the same loci, it suggests that these loci may be influencing incubation time independently of prion strain. This provides hope that it may be possible to identify human quantitative trait loci for prion incubation time using mouse models that may allow identification of at-risk individuals and the discovery of novel therapeutic targets. Electronic Publication     

Mapping of a FEB3 homologous febrile seizure locus on mouse chromosome 2 containing candidate genes Scn1a and Scn3a

Febrile seizures (FS) are the most common seizure type in children. Recurrent FS are a risk factor for developing temporal lobe epilepsy later in life and are known to have a strong genetic component. Experimental FS (eFS) can be elicited in mice by warm‐air induced hyperthermia. We used this model to screen the chromosome substitution strain (CSS) panel derived from C57BL/6J and A/J for FS susceptibility and identified C57BL/6J‐Chr2^A/NaJ (CSS2), as the strain with the strongest FS susceptibility phenotype. The aim of this study was to map FS susceptibility loci and select candidate genes on mouse chromosome 2. We generated an F₂ population by intercrossing the hybrids (F₁) that were derived from CSS2 and C57BL/6J mice. All CSS2‐F₂ individuals were genotyped and phenotyped for eFS susceptibility, and QTL analysis was performed. Candidate gene selection was based on bioinformatics analyses and differential brain expression between CSS2 and C57BL/6J strains determined by microarray analysis. Genetic mapping of the eFS susceptibility trait identified two significant loci: FS‐QTL2a (LOD‐score 3.6) and FS‐QTL2b (LOD‐score 6.2). FS‐QTL2a contained 44 genes expressed in the brain at post natal day 14. Four of these (Arl6ip6, Cytip, Fmnl2 Ifih1) contained a non‐synonymous SNP comparing CSS2 and C57BL/6J, six genes (March7, Nr4a2, Gpd2, Grb14, Scn1a, Scn3a) were differentially expressed between these strains. A region within FS‐QTL2a is homologous to the human FEB3 locus. The fact that we identify mouse FS‐QTL2a with high FEB3 homology is strong support for the validity of the eFS mouse model to study genetics of human FS.     

Mapping a mouse limbic seizure susceptibility locus on chromosome 10

Purpose: Mapping seizure susceptibility loci in mice provides a framework for identifying potentially novel candidate genes for human epilepsy. Using C57BL/6J × A/J chromosome substitution strains (CSS), we previously identified a locus on mouse chromosome 10 (Ch10) conferring susceptibility to pilocarpine, a muscarinic cholinergic agonist that models human temporal lobe epilepsy by inducing initial limbic seizures and status epilepticus (status), followed by hippocampal cell loss and delayed‐onset chronic spontaneous limbic seizures. Herein we report further genetic mapping of pilocarpine quantitative trait loci (QTLs) on Ch10. Methods: Seventy‐nine Ch10 F₂ mice were used to map QTLs for duration of partial status epilepticus and the highest stage reached in response to pilocarpine. Based on those results we created interval‐specific congenic lines to confirm and extend the results, using sequential rounds of breeding selectively by genotype to isolate segments of A/J Ch10 genome on a B6 background. Key Findings: Analysis of Ch10 F₂ genotypes and seizure susceptibility phenotypes identified significant, overlapping QTLs for duration of partial status and severity of pilocarpine‐induced seizures on distal Ch10. Interval‐specific Ch10 congenics containing the susceptibility locus on distal Ch10 also demonstrated susceptibility to pilocarpine‐induced seizures, confirming results from the F₂ mapping population and strongly supporting the presence of a QTL between rs13480781 (117.6 Mb) and rs13480832 (127.7 Mb). Significance: QTL mapping can identify loci that make a quantitative contribution to a trait, and eventually identify the causative DNA‐sequence polymorphisms. We have mapped a locus on mouse Ch10 for pilocarpine‐induced limbic seizures. Novel candidate genes identified in mice can be investigated in functional studies and tested for their role in human epilepsy.     

Systems genetic analysis of the effects of iron deficiency in mouse brain

Iron regulation in the brain is both necessary and highly complex. Too little or too much iron can compromise neurological function, yet we still do not know all of the regulatory processes. In our research, we seek to identify genes and gene networks underlying individual differences in brain iron regulation. To this end, we fed mice from 20+ inbred strains a diet low in iron from weaning to 4 months of age. At sacrifice, we measured iron content in the ventral midbrain (VMB). The VMB contains the substantia nigra, a region particularly vulnerable to iron imbalance. The results showed high, inter-strain variability in dietary iron reduction, from almost no loss to more than 40 % vs. control. When we performed quantitative trait loci (QTL) analysis, we observed a significant area on chromosome 2. Within this QTL, we selected glial high-affinity glutamate transporter 1 (Glt1) as the leading candidate. Expression of this gene is both correlated with VMB iron and is also cis-modulated by local sequence variants that segregate in the BXD family. VMB expression differences of Glt1 in six strains covary with differential susceptibility to VMB iron loss.     

Approach to depressogenic genes from genetic analyses of animal models]

Human depression or mood disorder is defined as a complex disease, making positional cloning of susceptibility genes a formidable task. We have undertaken genetic analyses of three different animal models for depression, comparing our results with advanced database resources. We first performed quantitative trait loci (QTL) analysis on two mouse models of "despair", namely, the forced swim test (FST) and tail suspension test (TST), and detected multiple chromosomal loci that control immobility time in these tests. Since one QTL detected on mouse chromosome 11 harbors the GABA A receptor subunit genes, we tested these genes for association in human mood disorder patients. We obtained significant associations of the alpha 1 and alpha 6 subunit genes with the disease, particularly in females. This result was striking, because we had previously detected an epistatic interaction between mouse chromosomes 11 and X that regulates immobility time in these animals. Next, we performed genome-wide expression analyses using a rat model of depression, learned helplessness (LH). We found that in the frontal cortex of LH rats, a disease implicated region, the LIM kinase 1 gene (Limk 1) showed greatest alteration, in this case down-regulation. By combining data from the QTL analysis of FST/TST and DNA microarray analysis of mouse frontal cortex, we identified adenylyl cyclase-associated CAP protein 1 (Cap 1) as another candidate gene for depression susceptibility. Both Limk 1 and Cap 1 are key players in the modulation of actin G-F conversion. In summary, our current study using animal models suggests disturbances of GABAergic neurotransmission and actin turnover as potential pathophysiologies for mood disorder.     

Genetic analysis reveals polygenic influences on iron, copper, and zinc in mouse hippocampus with neurobiological implications

Fe, Cu, and Zn are of widespread neurobiological importance, but must be regulated closely as too much or too little of these metals can have adverse effects on brain function. Recent evidence from nutritional models notes that the hippocampus is particularly vulnerable to Fe and Zn deficiencies. We recently performed a quantitative trait loci (QTL) analysis as a preliminary step in identifying genes that contribute to natural variation in hippocampal Fe, Cu, and Zn content. We used ICP-MS to measure the concentrations of these metals in 120-day-old mice from 30 strains of the BXD/TY panel. The BXD/Ty recombinant inbred strain panel is well-suited for complex trait analysis, as all strains are genotyped with a dense marker set and have been phenotyped extensively for neurobehavioral traits and hippocampal gene expression. We observed a wide-range of hippocampal Fe, Cu, and Zn concentrations across the BXD strains. These concentrations were related to systemic Fe status, but not to Fe, Cu, and Zn elsewhere in the brain. The three metals also showed strong covariance, suggestive of overlap in their regulatory pathways. We identified two QTL, on chromosomes 14 and 9, most strongly associated with Cu but also suggestively associated with Fe (chr. 14) and Zn (chr. 9). We also performed genetic correlational analyses with existing data on these strains and revealed associations with cognitive, anxiety-related, and alcohol-related phenotypes. Covariance of these metals with gene expression is also discussed. This work shows that hippocampal Fe, Cu, and Zn are under polygenic influence and that trace metal regulation is associated with hippocampus-related behaviors. Future work will elucidate the genes underlying the two QTL identified, to aid in identifying homologous genetic variants in human populations, which may underlie altered trace metal homeostasis and related neurological disease.     

Quantitative trait loci for novelty/stress-induced locomotor activation in recombinant inbred (RI) and recombinant congenic (RC) strains of mice

The objective of the present study was to map and compare quantitative trait loci (QTLs) for an anxiety-related trait (novelty/stress-induced activation) in the AXB/BXA recombinant inbred (RI) and AcB/BcA recombinant congenic (RC) strains of mice derived from the A/J and C57BL/6J inbred progenitor strains. Activational responses to a novel open field (OF) were measured under identical stressful conditions (no prior handling or exposure to testing procedures) in both the RI and RC strains. Naive male and female mice were weighed, injected with IP saline and locomotor activity was monitored in a computerized OF apparatus for 15 min. Measures obtained from this experimental design included: (1) total activity scores, (2) time course of response (5 min time blocks over the 15 min session). Data for the RI strains were subjected to a QTL analysis using composite interval mapping. Significant loci were identified on chr 5 (D5Mit356, 41 cM), chr 8 (D8Mit305, 37 cM) and chr 14 (D14Mit36, 6 3cM). Single locus association analysis of the AcB/BcA RC strains identified 15 putative regions, 7 of which overlapped regions independently mapped in the RI strains on chr 1 (58.5-63.1cM), chr 4 (21.9-28.6 cM), chr 5 (19-45 & 74-86 cM), chr 6 (0.5-20.4cM), chr 9 (15-38 cM), chr 13 (47cM) and chr 19 (47cM). The loci identified on chr 5 near D5Mit356 (41cM) in both the AXB/BXA RIS and AcB/BcA RCS maps to a region containing the genes for several GABA(A) receptor subunits. Additionally, the present study provides further confirmation of a frequently identified QTL on chromosome 1. The results are discussed in the context of previous QTL studies of anxiety-related traits that have used genetic crosses that include the A or B6 progenitors.     

Role of an Atypical Cadherin Gene,Cdh23 in Prepulse Inhibition,and Implication of CDH23 in Schizophrenia

We previously identified quantitative trait loci (QTL) for prepulse inhibition (PPI), an endophenotype of schizophrenia, on mouse chromosome 10 and reported Fabp7 as a candidate gene from an analysis of F2 mice from inbred strains with high (C57BL/6N; B6) and low (C3H/HeN; C3H) PPI levels. Here, we reanalyzed the previously reported QTLs with increased marker density. The highest logarithm of odds score (26.66) peaked at a synonymous coding and splice-site variant, c.753G>A (rs257098870), in the Cdh23 gene on chromosome 10; the c.753G (C3H) allele showed a PPI-lowering effect. Bayesian multiple QTL mapping also supported the same variant with a posterior probability of 1. Thus, we engineered the c.753G (C3H) allele into the B6 genetic background, which led to dampened PPI. We also revealed an e-QTL (expression QTL) effect imparted by the c.753G>A variant for the Cdh23 expression in the brain. In a human study, a homologous variant (c.753G>A; rs769896655) in CDH23 showed a nominally significant enrichment in individuals with schizophrenia. We also identified multiple potentially deleterious CDH23 variants in individuals with schizophrenia. Collectively, the present study reveals a PPI-regulating Cdh23 variant and a possible contribution of CDH23 to schizophrenia susceptibility.     

Genome-wide linkage scan of quantitative traits representing symptom dimensions in multiplex schizophrenia families

Symptom dimensions of schizophrenia are likely to be the intermediate phenotypes under the control of disease-susceptibility genes, or separate traits related to disease-modifier genes. This study aimed to identify chromosomal loci linked to symptom dimensions of schizophrenia through genome-wide quantitative trait locus (QTL) linkage analysis. The study subjects consisted of 56 families with 183 members including 123 affected individuals. Symptom evaluations were performed on lifetime basis. Through principal component factor analysis, eight quantitative phenotypes representing symptom dimensions were identified. Genotyping was done for 6008 SNP markers, and genome-wide QTL linkage analysis was performed. No symptom dimension showed a significant linkage attaining genome-wide empirical thresholds. We observed seven regions yielding linkage signals attaining genome-wide empirical thresholds for suggestive linkage (NPL Z score=2.78–3.49); chromosome 15q26.1 for ‘non-paranoid delusion factor’, 2p24.3 and 7q31.1 for ‘prodromal impairment factor’, 1q32.1, 9p21.3, and 9q31.2 for ‘negative symptom factor’, and 10p13 for ‘disorganization factor’. Among these loci, chromosome 2p24.3 and 1q32.1 overlap with susceptibility loci of schizophrenia identified in our previous linkage studies. This study suggests the existence of genetic loci related to various clinical features of schizophrenia. Further genetic analyses for these dimensional phenotypes are warranted.     

Quantitative trait locus analysis of nonverbal communication in autism spectrum disorder

Autism spectrum disorder (ASD) is a neurodevelopmental syndrome marked by impairments in social interactive functioning and communication skills, and the presence of repetitive and restrictive behaviors. Twin and linkage studies provide evidence that ASD is heritable and genetically complex. Genetic analyses of familial quantitative traits in those with ASD may help to reveal underlying risk genes. We report a quantitative trait locus (QTL) analysis of nonverbal communication (NVC) in 228 families from the autism genetics resource exchange (AGRE) ascertained for at least two siblings with ASD. QTL at 1p13-q12, 4q21-25, 7q35, 8q23-24, and 16p12-13 indicate that genes at these loci may contribute to the variation in NVC among those with ASD. Using the criteria of Lander and Kruglyak, the QTL at 1p13-q12 is 'suggestive', while the other four are 'possible'. To assess whether these QTL are likely to harbor genes contributing specifically to the deficits in NVC, linkage analysis of ASD sibships with the most severe NVC scores was conducted. The sibships were identified by ordered-subset analyses (OSA), and families with the most severe NVC scores displayed lod scores of 3.4 at 8q23-24 and 3.8 at 16p12-13, indicating that these two regions are likely to harbor gene(s) contributing to ASD by predisposing to deficits in NVC.     

Genetic risk mechanisms of posttraumatic stress disorder in the human brain

Posttraumatic stress disorder (PTSD) follows exposure to a traumatic event in susceptible individuals. Recently, genome‐wide association studies have identified a number of genetic sequence variants that are associated with the risk of developing PTSD. To follow up on identifying the molecular mechanisms of these risk variants, we performed genotype to RNA sequencing–derived quantitative expression (whole gene, exon, and exon junction levels) analysis in the dorsolateral prefrontal cortex (DLPFC) of normal postmortem human brains. We further investigated genotype–gene expression associations within the amygdala in a smaller independent RNA sequencing (Genotype‐Tissue Expression [GTEx]) dataset. Our DLPFC analyses identified significant expression quantitative trait loci (eQTL) associations for a “candidate” PTSD risk SNP rs363276 and the expression of two genes: SLC18A2 and PDZD8, where the PTSD risk/minor allele T was associated with significantly lower levels of gene expression for both genes, in the DLPFC. These eQTL associations were independently confirmed in the amygdala from the GTEx database. Rs363276 “T” carriers also showed significantly increased activity in the amygdala during an emotional face‐matching task in healthy volunteers. Taken together, our preliminary findings in normal human brains represent a tractable approach to identify mechanisms by which genetic variants potentially increase an individual's risk for developing PTSD. © 2016 Wiley Periodicals, Inc.     

A QTL on rat chromosome 7 modulates prepulse inhibition, a neuro-behavioral trait of ADHD, in a Lewis x SHR intercross

Background  

Attention deficit hyperactivity disorder (ADHD) is a complex neuropsychiatric disorder with a substantial genetic component. The Spontaneously Hypertensive Rats (SHR), considered as a good animal model of ADHD, also show less anxiety-like behaviors than Lewis (LEW) rats. The use of these inbred rat strains led us to the mapping of two quantitative trait loci (QTL), named Ofil1 (on chromosome 4) and Ofil2 (on chromosome 7), related to locomotion in the central and aversive area of an open field. Herein, we examined whether LEW and SHR rats differ in the acoustic startle reflex, a test used to study the neurobiology of anxiety, and in the prepulse inhibition of the startle response, which is known to be impaired in ADHD patients. The effect of the two aforementioned loci on these behavioral responses was also studied.     

Creutzfeldt-Jakob disease and scrapie prions

Creutzfeldt-Jakob disease, kuru, and Gerstmann-Str?ussler syndrome are transmissible degenerative diseases of the central nervous system caused by novel infectious pathogens designated prions. Scrapie is a neurodegenerative disease of sheep and goats and is also caused by prions. Experimental scrapie has been extensively studied in hamsters and mice. The scrapie prion protein (PrPSc) is the only component of the infectious scrapie prion identified, to date. Scrapie infectivity and PrPSc copartition into membranes, rods, and liposomes raising the possibility that only PrPSc might be required for infection; however, a second component such as a small nucleic acid cannot be eliminated. PrPSc is encoded by a single copy cellular gene and not by a hypothetical nucleic acid within purified prion preparations. Normal, uninfected cells express the cellular prion protein (PrPc). Both PrPSc and PrPc appear to be translated from the same 2.1-kb mRNA. The N-terminal amino acid sequences of hamster PrPC and PrPSc are identical; both correspond to that predicted by the translated prion protein (PrP) gene sequence. While the chemical difference between PrPc and PrPSc remains unknown, the organization of the PrP gene argues that it results from a posttranslational event. Six posttranslational modifications of both PrP isoforms have been identified: (1) cleavage of an N-terminal signal peptide, (2) an intramolecular disulfide bond, (3) an N-linked oligosaccharide attached to Asn 181, (4) a second oligosaccharide attached to Asn 197, (5) cleavage of a C-terminal hydrophobic peptide, and (6) a phosphatidylinositol glycolipid attached to the C-terminus. The mouse PrP gene is on chromosome 2 and is linked to a gene controlling the scrapie incubation time (Prn-i). PrP genes from inbred mice with short and long incubation times differ by two amino acids, a finding consistent with but not proving that PrP modulates susceptibility to scrapie. PrPSc stimulation of a posttranslational process which converts PrPc or its precursor into PrPSc is one possible mechanism for prion replication. This is consistent with observations showing that human prion diseases are manifest as infectious, sporadic and genetic disorders.     

Cathepsin D exon 2 polymorphism associated with general intelligence in a healthy older population

General intelligence is a heritable trait that is a risk factor for both the onset of dementia and the rate of cognitive decline in community-dwelling older persons. Previous studies screening for quantitative trait loci (QTLs) that influence general intelligence in healthy individuals have identified four loci, two of which are located within the genes insulin-like growth factor 2 receptor (IGF2R) and the Msx1 homeobox. Here, we report the finding of another QTL associated with general intelligence that is located within exon 2 of the cathepsin D (CTSD) gene. A group of 767 healthy adults with a follow-up period of over 15 years have been analyzed for cross-sectional and longitudinal trends in cognitive change using the Heim intelligence test score (AH4-1). We observed a significant association (P = 0.01) between a functional C > T (Ala > Val) transition within exon 2 of the CTSD gene that increases the secretion of pro-CTSD from the cell, and the AH4-1 score at initial testing on entry to the longitudinal study. Interestingly, CTSD is transported by IGF2R from the trans Golgi network to the lysosome.     

Quantitative trait loci modulate ventricular size in the mouse brain

Cerebral ventricular size in humans varies significantly. Abnormal enlargement of the ventricles has been associated with schizophrenia, and hydrocephalus can lead to serious cognitive and motor deficiencies in humans and animals. In this study, we mapped quantitative trait loci (QTLs) modulating cerebroventricular size in mice. We hypothesized that genes underlying hydrocephalus might also modulate normal variation in ventricular size. By using digital images of mouse brain sections and stereological techniques, we estimated the volume of the combined lateral and third ventricles, as well as the volume of the entire brain, in 228 AXB and BXA recombinant inbred mice and their parent strains (A/J and C57BL/6J). Ventricle size, expressed as percentage of brain volume, is a heritable trait (h(2) = 0.32). We detected a major QTL controlling variance in volume on chromosome (Chr) 8 near the markers D8Mit94 and D8Mit189. We also detected a strong epistatic interaction affecting ventricular volume between loci on Chr 4 (near D4Mit237 and D4Mit214) and on Chr 7 (D7Mit178 and D7Mit191). These three QTLs, labeled Vent8a, Vent4b, and Vent7c, are close to genes that have been previously implicated in hydrocephalus.     

Further dissection of a genomic locus associated with behavioral activity in the Wistar-Kyoto hyperactive rat,an animal model of hyperkinesis

Molecular genetic studies of attention-deficit hyperactivity disorder (ADHD) are a major focus of current research since this syndrome has been shown to be highly heritable.(1) Our approach has been to search for quantitative trait loci (QTL) in a genetic animal model of hyperkinesis, the Wistar-Kyoto hyperactive (WKHA) rat, by a whole-genome scan analysis. In a previous article, we reported the detection of a major QTL associated with behavioral activity in an F2 cross between WKHA and Wistar-Kyoto (WKY) rat strains.(2) Here, we extend our analysis of this cross by adding new genetic markers, now defining a 10 cM interval on rat chromosome 8 associated with ambulatory and exploratory activities. Then we present a replication of this QTL detection, at least for exploratory activity, by a new genetic mapping analysis of an activity QTL in an F2 cross between the WKHA and Brown Norway (BN) rat strains. Overall, the results provide compelling evidence for the presence of gene(s) influencing activity at this locus. The QTL interval has been refined such that the human orthologous region could be defined and tested in human populations for association with ADHD. Ultimately, the improved dissection of this genomic locus should allow the identification of the causal genes.     

Identification of female-specific QTLs affecting an emotionality-related behavior in rats.

The influence of genetic factors on psychological traits and disorders has been repeatedly demonstrated; however, the molecular mechanisms underlying such an influence remain largely unknown. Anxiety-related disorders constitute the most common class of mental disorder in humans, with women being diagnosed far more frequently than men. A better understanding of the genetic and gender-related mechanisms mediating anxiety traits should enable the development of more rational methods for preventing and treating anxiety disorders. In this study we have aimed to identify, for the first time, quantitative trait loci (QTL) influencing anxiety/emotionality-related traits in rats. To this end, two strains-Lewis (LEW) and Spontaneously Hypertensive Rats (SHR)-that differ for several behavioral measures of anxiety/emotionality were intercrossed. A QTL analysis of the F2 population revealed suggestive loci for various traits, including behaviors in the elevated plus-maze and blood pressure. In addition, one major QTL explaining 50.4% of the total variance (LOD = 7.22) was identified on chromosome 4 for the locomotion in the central and aversive area of the open field. Two other relevant QTLs have been recently mapped near this chromosomic region in the rat, which also harbors Tac1r, the gene encoding for the substance P receptor. Our major QTL affected females but not males and its effect depended on the type of cross (LEW or SHR grandmothers). The present results reveal a complex genetic basis underlying emotional behaviors and they confirm the existence of interactions between genetic factors and sex for this kind of trait. Further investigation of the loci identified herein may give clues to the pathophysiology of psychiatric disorders such as anxiety-related ones.