Mapping the gene for autosomal dominant restless legs syndrome in an Irish family

Restless Legs Syndrome (RLS) is a common neurological disorder affecting nearly 15% of the general population. Ironically, RLS can be described as the most common condition one has never heard of. It is usually characterised by uncomfortable, unpleasant sensations in the lower limbs inducing an uncontrollable desire to move the legs. RLS exhibits a circadian pattern with symptoms present predominantly in the evening or at night, thus leading to sleep disruption and daytime somnolence. RLS is generally classified into primary (idiopathic) and secondary (symptomatic) forms. Primary RLS includes sporadic and familial cases of which the age of onset is usually less than 45 years and progresses slowly with a female to male ratio of 2:1. Secondary forms often occur as a complication of another health condition, such as iron deficiency or thyroid dysfunction. The age of onset is usually over 45 years, with an equal male to female ratio and more rapid progression. Ekbom described the familial component of the disorder in 1945 and since then many studies have been published on the familial forms of the disorder. Molecular genetic studies have so far identified ten loci (5q, 12q, 14p, 9p, 20p, 16p, 19p, 4q, 17p). No specific gene within these loci has been identified thus far. Association mapping has highlighted a further five areas of interest. RLS6 has been found to be associated with SNPs in the BTBD9 gene. Four other variants were found within intronic and intergenic regions of MEIS1, MAP2K5/LBXCOR1, PTPRD and NOS1. The pathophysiology of RLS is complex and remains to be fully elucidated. Conditions associated with secondary RLS, such as pregnancy or end-stage renal disease, are characterised by iron deficiency, which suggests that disturbed iron homeostasis plays a role. Dopaminergic dysfunction in subcortical systems also appears to play a central role. An ongoing study within the Department of Pathology (University College Cork) is investigating the genetic characteristics of RLS in Irish families. A three generation RLS pedigree RLS3002 consisting of 11 affected and 7 unaffected living family members was recruited. The family had been examined for four of the known loci (5q, 12q, 14p and 9p) (Abdulrahim 2008). The aim of this study was to continue examining this Irish RLS pedigree for possible linkage to the previously described loci and associated regions. Using informative microsatellite markers linkage was excluded to the loci on 5q, 12q, 14p, 9p, 20p, 16p, 19p, 4q, 17p and also within the regions reported to be associated with RLS. This suggested the presence of a new unidentified locus. A genome-wide scan was performed using two microsatellite marker screening sets (Research Genetics Inc. Mapping set and the Applied Biosystems Linkage mapping set version 2.5). Linkage analysis was conducted under an autosomal dominant model with a penetrance of 95% and an allele frequency of 0.01. A maximum LOD score of 3.59 at θ=0.00 for marker D19S878 indicated significant linkage on chromosome 19p. Haplotype analysis defined a genetic region of 6.57 cM on chromosome 19p13.3, corresponding to 2.5 Mb. There are approximately 100 genes annotated within the critical region. Sequencing of two candidate genes, KLF16 and GAMT, selected on the assumed pathophysiology of RLS, did not identify any sequence variant. This study provides evidence of a novel RLS locus in an Irish pedigree, thus supporting the picture of RLS as a genetically heterogeneous trait.

Alpha-fetoprotein controls female fertility and prenatal development of the gonadotropin-releasing hormone pathway through an antiestrogenic action

It has been shown previously that female mice homozygous for an alpha-fetoprotein (AFP) null allele are sterile as a result of anovulation, probably due to a defect in the hypothalamic-pituitary axis. Here we show that these female mice exhibit specific anomalies in the expression of numerous genes in the pituitary, including genes involved in the gonadotropin-releasing hormone pathway, which are underexpressed. In the hypothalamus, the gonadotropin-releasing hormone gene, Gnrh1, was also found to be down-regulated. However, pituitary gene expression could be normalized and fertility could be rescued by blocking prenatal estrogen synthesis using an aromatase inhibitor. These results show that AFP protects the developing female brain from the adverse effects of prenatal estrogen exposure and clarify a long-running debate on the role of this fetal protein in brain sexual differentiation.

Traumatic brain injury exacerbates neurodegenerative pathology: improvement with an apolipoprotein E-based therapeutic.

Cognitive impairment is common following traumatic brain injury (TBI), and neuroinflammatory mechanisms may predispose to the development of neurodegenerative disease. Apolipoprotein E (apoE) polymorphisms modify neuroinflammatory responses, and influence both outcome from acute brain injury and the risk of developing neurodegenerative disease. We demonstrate that TBI accelerates neurodegenerative pathology in double-transgenic animals expressing the common human apoE alleles and mutated amyloid precursor protein, and that pathology is exacerbated in the presence of the apoE4 allele. The administration of an apoE-mimetic peptide markedly reduced the development of neurodegenerative pathology in mice homozygous for apoE3 as well as apoE3/E4 heterozygotes. These results demonstrate that TBI accelerates the cardinal neuropathological features of neurodegenerative disease, and establishes the potential for apoE mimetic therapies in reducing pathology associated with neurodegeneration.

Host determinants of HIV-1 control in African Americans.

We performed a whole-genome association study of human immunodeficiency virus type 1 (HIV-1) set point among a cohort of African Americans (n = 515), and an intronic single-nucleotide polymorphism (SNP) in the HLA-B gene showed one of the strongest associations. We use a subset of patients to demonstrate that this SNP reflects the effect of the HLA-B*5703 allele, which shows a genome-wide statistically significant association with viral load set point (P = 5.6 x 10(-10)). These analyses therefore confirm a member of the HLA-B*57 group of alleles as the most important common variant that influences viral load variation in African Americans, which is consistent with what has been observed for individuals of European ancestry, among whom the most important common variant is HLA-B*5701.

BMP signaling in the development of the mouse esophagus and forestomach.

The stratification and differentiation of the epidermis are known to involve the precise control of multiple signaling pathways. By contrast, little is known about the development of the mouse esophagus and forestomach, which are composed of a stratified squamous epithelium. Based on prior work in the skin, we hypothesized that bone morphogenetic protein (BMP) signaling is a central player. To test this hypothesis, we first used a BMP reporter mouse line harboring a BRE-lacZ allele, along with in situ hybridization to localize transcripts for BMP signaling components, including various antagonists. We then exploited a Shh-Cre allele that drives recombination in the embryonic foregut epithelium to generate gain- or loss-of-function models for the Bmpr1a (Alk3) receptor. In gain-of-function (Shh-Cre;Rosa26(CAG-loxpstoploxp-caBmprIa)) embryos, high levels of ectopic BMP signaling stall the transition from simple columnar to multilayered undifferentiated epithelium in the esophagus and forestomach. In loss-of-function experiments, conditional deletion of the BMP receptor in Shh-Cre;Bmpr1a(flox/flox) embryos allows the formation of a multilayered squamous epithelium but this fails to differentiate, as shown by the absence of expression of the suprabasal markers loricrin and involucrin. Together, these findings suggest multiple roles for BMP signaling in the developing esophagus and forestomach.

Rare variants create synthetic genome-wide associations.

Genome-wide association studies (GWAS) have now identified at least 2,000 common variants that appear associated with common diseases or related traits (http://www.genome.gov/gwastudies), hundreds of which have been convincingly replicated. It is generally thought that the associated markers reflect the effect of a nearby common (minor allele frequency >0.05) causal site, which is associated with the marker, leading to extensive resequencing efforts to find causal sites. We propose as an alternative explanation that variants much less common than the associated one may create "synthetic associations" by occurring, stochastically, more often in association with one of the alleles at the common site versus the other allele. Although synthetic associations are an obvious theoretical possibility, they have never been systematically explored as a possible explanation for GWAS findings. Here, we use simple computer simulations to show the conditions under which such synthetic associations will arise and how they may be recognized. We show that they are not only possible, but inevitable, and that under simple but reasonable genetic models, they are likely to account for or contribute to many of the recently identified signals reported in genome-wide association studies. We also illustrate the behavior of synthetic associations in real datasets by showing that rare causal mutations responsible for both hearing loss and sickle cell anemia create genome-wide significant synthetic associations, in the latter case extending over a 2.5-Mb interval encompassing scores of "blocks" of associated variants. In conclusion, uncommon or rare genetic variants can easily create synthetic associations that are credited to common variants, and this possibility requires careful consideration in the interpretation and follow up of GWAS signals.

Serotonin transporter genotype modulates social reward and punishment in rhesus macaques

Background: Serotonin signaling influences social behavior in both human and nonhuman primates. In humans, variation upstream of the promoter region of the serotonin transporter gene (5-HTTLPR) has recently been shown to influence both behavioral measures of social anxiety and amygdala response to social threats. Here we show that length polymorphisms in 5-HTTLPR predict social reward and punishment in rhesus macaques, a species in which 5-HTTLPR variation is analogous to that of humans. Methodology/Principal Findings: In contrast to monkeys with two copies of the long allele (L/L), monkeys with one copy of the short allele of this gene (S/L) spent less time gazing at face than non-face images, less time looking in the eye region of faces, and had larger pupil diameters when gazing at photos of a high versus low status male macaques. Moreover, in a novel primed gambling task, presentation of photos of high status male macaques promoted risk-aversion in S/L monkeys but promoted risk-seeking in L/L monkeys. Finally, as measured by a "pay-per-view" task, S/L monkeys required juice payment to view photos of high status males, whereas L/L monkeys sacrificed fluid to see the same photos. Conclusions/Significance: These data indicate that genetic variation in serotonin function contributes to social reward and punishment in rhesus macaques, and thus shapes social behavior in humans and rhesus macaques alike. © 2009 Watson et al.

Structure, function, and phylogeny of the mating locus in the Rhizopus oryzae complex.

The Rhizopus oryzae species complex is a group of zygomycete fungi that are common, cosmopolitan saprotrophs. Some strains are used beneficially for production of Asian fermented foods but they can also act as opportunistic human pathogens. Although R. oryzae reportedly has a heterothallic (+/-) mating system, most strains have not been observed to undergo sexual reproduction and the genetic structure of its mating locus has not been characterized. Here we report on the mating behavior and genetic structure of the mating locus for 54 isolates of the R. oryzae complex. All 54 strains have a mating locus similar in overall organization to Phycomyces blakesleeanus and Mucor circinelloides (Mucoromycotina, Zygomycota). In all of these fungi, the minus (-) allele features the SexM high mobility group (HMG) gene flanked by an RNA helicase gene and a TP transporter gene (TPT). Within the R. oryzae complex, the plus (+) mating allele includes an inserted region that codes for a BTB/POZ domain gene and the SexP HMG gene. Phylogenetic analyses of multiple genes, including the mating loci (HMG, TPT, RNA helicase), ITS1-5.8S-ITS2 rDNA, RPB2, and LDH genes, identified two distinct groups of strains. These correspond to previously described sibling species R. oryzae sensu stricto and R. delemar. Within each species, discordant gene phylogenies among multiple loci suggest an outcrossing population structure. The hypothesis of random-mating is also supported by a 50:50 ratio of plus and minus mating types in both cryptic species. When crossed with tester strains of the opposite mating type, most isolates of R. delemar failed to produce zygospores, while isolates of R. oryzae produced sterile zygospores. In spite of the reluctance of most strains to mate in vitro, the conserved sex locus structure and evidence for outcrossing suggest that a normal sexual cycle occurs in both species.

Assessment of LD matrix measures for the analysis of biological pathway association.

Complex diseases will have multiple functional sites, and it will be invaluable to understand the cross-locus interaction in terms of linkage disequilibrium (LD) between those sites (epistasis) in addition to the haplotype-LD effects. We investigated the statistical properties of a class of matrix-based statistics to assess this epistasis. These statistical methods include two LD contrast tests (Zaykin et al., 2006) and partial least squares regression (Wang et al., 2008). To estimate Type 1 error rates and power, we simulated multiple two-variant disease models using the SIMLA software package. SIMLA allows for the joint action of up to two disease genes in the simulated data with all possible multiplicative interaction effects between them. Our goal was to detect an interaction between multiple disease-causing variants by means of their linkage disequilibrium (LD) patterns with other markers. We measured the effects of marginal disease effect size, haplotype LD, disease prevalence and minor allele frequency have on cross-locus interaction (epistasis). In the setting of strong allele effects and strong interaction, the correlation between the two disease genes was weak (r=0.2). In a complex system with multiple correlations (both marginal and interaction), it was difficult to determine the source of a significant result. Despite these complications, the partial least squares and modified LD contrast methods maintained adequate power to detect the epistatic effects; however, for many of the analyses we often could not separate interaction from a strong marginal effect. While we did not exhaust the entire parameter space of possible models, we do provide guidance on the effects that population parameters have on cross-locus interaction.

Genome-wide scan of copy number variation in late-onset Alzheimer's disease.

Alzheimer's disease is a complex and progressive neurodegenerative disease leading to loss of memory, cognitive impairment, and ultimately death. To date, six large-scale genome-wide association studies have been conducted to identify SNPs that influence disease predisposition. These studies have confirmed the well-known APOE epsilon4 risk allele, identified a novel variant that influences disease risk within the APOE epsilon4 population, found a SNP that modifies the age of disease onset, as well as reported the first sex-linked susceptibility variant. Here we report a genome-wide scan of Alzheimer's disease in a set of 331 cases and 368 controls, extending analyses for the first time to include assessments of copy number variation. In this analysis, no new SNPs show genome-wide significance. We also screened for effects of copy number variation, and while nothing was significant, a duplication in CHRNA7 appears interesting enough to warrant further investigation.

Effects of neuronal PIK3C3/Vps34 deletion on autophagy and beyond.

PIK3C3/Vps34 plays important roles in the endocytic and autophagic pathways, both of which are essential for maintaining neuronal integrity. However, it is unclear how inactivating PIK3C3 may affect neuronal endosomal versus autophagic processes in vivo. We generated a conditional null allele of the Pik3c3 gene in mouse, and specifically deleted it in postmitotic sensory neurons. Subsequent analyses reveal several interesting and surprising findings.

Associations between Intake of Folate, Methionine, and Vitamins B-12, B-6 and Prostate Cancer Risk in American Veterans.

Prostate cancer (PC) is the second leading cause of cancer death in men. Recent reports suggest that excess of nutrients involved in the one-carbon metabolism pathway increases PC risk; however, empirical data are lacking. Veteran American men (272 controls and 144 PC cases) who attended the Durham Veteran American Medical Center between 2004-2009 were enrolled into a case-control study. Intake of folate, vitamin B12, B6, and methionine were measured using a food frequency questionnaire. Regression models were used to evaluate the association among one-carbon cycle nutrients, MTHFR genetic variants, and prostate cancer. Higher dietary methionine intake was associated with PC risk (OR = 2.1; 95%CI 1.1-3.9) The risk was most pronounced in men with Gleason sum <7 (OR = 2.75; 95%CI 1.32- 5.73). The association of higher methionine intake and PC risk was only apparent in men who carried at least one MTHFR A1298C allele (OR = 6.7; 95%CI = 1.6-27.8), compared to MTHFR A1298A noncarrier men (OR = 0.9; 95%CI = 0.24-3.92) (p-interaction = 0.045). There was no evidence for associations between B vitamins (folate, B12, and B6) and PC risk. Our results suggest that carrying the MTHFR A1298C variants modifies the association between high methionine intake and PC risk. Larger studies are required to validate these findings.

Independent evolutionary origins of landlocked alewife populations and rapid parallel evolution of phenotypic traits.

Alewife, Alosa pseudoharengus, populations occur in two discrete life-history variants, an anadromous form and a landlocked (freshwater resident) form. Landlocked populations display a consistent pattern of life-history divergence from anadromous populations, including earlier age at maturity, smaller adult body size, and reduced fecundity. In Connecticut (USA), dams constructed on coastal streams separate anadromous spawning runs from lake-resident landlocked populations. Here, we used sequence data from the mtDNA control region and allele frequency data from five microsatellite loci to ask whether coastal Connecticut landlocked alewife populations are independently evolved from anadromous populations or whether they share a common freshwater ancestor. We then used microsatellite data to estimate the timing of the divergence between anadromous and landlocked populations. Finally, we examined anadromous and landlocked populations for divergence in foraging morphology and used divergence time estimates to calculate the rate of evolution for foraging traits. Our results indicate that landlocked populations have evolved multiple times independently. Tests of population divergence and estimates of gene flow show that landlocked populations are genetically isolated, whereas anadromous populations exchange genes. These results support a 'phylogenetic raceme' model of landlocked alewife divergence, with anadromous populations forming an ancestral core from which landlocked populations independently diverged. Divergence time estimates suggest that landlocked populations diverged from a common anadromous ancestor no longer than 5000 years ago and perhaps as recently as 300 years ago, depending on the microsatellite mutation rate assumed. Examination of foraging traits reveals landlocked populations to have significantly narrower gapes and smaller gill raker spacings than anadromous populations, suggesting that they are adapted to foraging on smaller prey items. Estimates of evolutionary rates (in haldanes) indicate rapid evolution of foraging traits, possibly in response to changes in available resources.

A functional polymorphism in the 5HTR2C gene associated with stress responses also predicts incident cardiovascular events.

Previously we have shown that a functional nonsynonymous single nucleotide polymorphism (rs6318) of the 5HTR2C gene located on the X-chromosome is associated with hypothalamic-pituitary-adrenal axis response to a stress recall task, and with endophenotypes associated with cardiovascular disease (CVD). These findings suggest that individuals carrying the rs6318 Ser23 C allele will be at higher risk for CVD compared to Cys23 G allele carriers. The present study examined allelic variation in rs6318 as a predictor of coronary artery disease (CAD) severity and a composite endpoint of all-cause mortality or myocardial infarction (MI) among Caucasian participants consecutively recruited through the cardiac catheterization laboratory at Duke University Hospital (Durham, NC) as part of the CATHGEN biorepository. Study population consisted of 6,126 Caucasian participants (4,036 [65.9%] males and 2,090 [34.1%] females). A total of 1,769 events occurred (1,544 deaths and 225 MIs; median follow-up time = 5.3 years, interquartile range = 3.3-8.2). Unadjusted Cox time-to-event regression models showed, compared to Cys23 G carriers, males hemizygous for Ser23 C and females homozygous for Ser23C were at increased risk for the composite endpoint of all-cause death or MI: Hazard Ratio (HR) = 1.47, 95% confidence interval (CI) = 1.17, 1.84, p = .0008. Adjusting for age, rs6318 genotype was not related to body mass index, diabetes, hypertension, dyslipidemia, smoking history, number of diseased coronary arteries, or left ventricular ejection fraction in either males or females. After adjustment for these covariates the estimate for the two Ser23 C groups was modestly attenuated, but remained statistically significant: HR = 1.38, 95% CI = 1.10, 1.73, p = .005. These findings suggest that this functional polymorphism of the 5HTR2C gene is associated with increased risk for CVD mortality and morbidity, but this association is apparently not explained by the association of rs6318 with traditional risk factors or conventional markers of atherosclerotic disease.

Mutations in NGLY1 cause an inherited disorder of the endoplasmic reticulum-associated degradation pathway.

PURPOSE: The endoplasmic reticulum-associated degradation pathway is responsible for the translocation of misfolded proteins across the endoplasmic reticulum membrane into the cytosol for subsequent degradation by the proteasome. To define the phenotype associated with a novel inherited disorder of cytosolic endoplasmic reticulum-associated degradation pathway dysfunction, we studied a series of eight patients with deficiency of N-glycanase 1. METHODS: Whole-genome, whole-exome, or standard Sanger sequencing techniques were employed. Retrospective chart reviews were performed in order to obtain clinical data. RESULTS: All patients had global developmental delay, a movement disorder, and hypotonia. Other common findings included hypolacrima or alacrima (7/8), elevated liver transaminases (6/7), microcephaly (6/8), diminished reflexes (6/8), hepatocyte cytoplasmic storage material or vacuolization (5/6), and seizures (4/8). The nonsense mutation c.1201A>T (p.R401X) was the most common deleterious allele. CONCLUSION: NGLY1 deficiency is a novel autosomal recessive disorder of the endoplasmic reticulum-associated degradation pathway associated with neurological dysfunction, abnormal tear production, and liver disease. The majority of patients detected to date carry a specific nonsense mutation that appears to be associated with severe disease. The phenotypic spectrum is likely to enlarge as cases with a broader range of mutations are detected.