A high-density association screen of 155 ion transport genes for involvement with common migraine

The clinical overlap between monogenic Familial Hemiplegic Migraine (FHM) and common migraine subtypes, and the fact that all three FHM genes are involved in the transport of ions, suggest that ion transport genes may underlie susceptibility to common forms of migraine. To test this leading hypothesis, we examined common variation in 155 ion transport genes using 5257 single nucleotide polymorphisms (SNPs) in a Finnish sample of 841 unrelated migraine with aura cases and 884 unrelated non-migraine controls. The top signals were then tested for replication in four independent migraine case-control samples from the Netherlands, Germany and Australia, totalling 2835 unrelated migraine cases and 2740 unrelated controls. SNPs within 12 genes (KCNB2, KCNQ3, CLIC5, ATP2C2, CACNA1E, CACNB2, KCNE2, KCNK12, KCNK2, KCNS3, SCN5A and SCN9A) with promising nominal association (0.00041 < P < 0.005) in the Finnish sample were selected for replication. Although no variant remained significant after adjusting for multiple testing nor produced consistent evidence for association across all cohorts, a significant epistatic interaction between KCNB2 SNP rs1431656 (chromosome 8q13.3) and CACNB2 SNP rs7076100 (chromosome 10p12.33) (pointwise P = 0.00002; global P = 0.02) was observed in the Finnish case-control sample. We conclude that common variants of moderate effect size in ion transport genes do not play a major role in susceptibility to common migraine within these European populations, although there is some evidence for epistatic interaction between potassium and calcium channel genes, KCNB2 and CACNB2. Multiple rare variants or trans-regulatory elements of these genes are not ruled out.

A genome-wide linkage scan provides evidence for both new and previously reported loci influencing common migraine

Latent class analysis was performed on migraine symptom data collected in a Dutch population sample (N = 12,210, 59% female) in order to obtain empirical groupings of individuals suffering from symptoms of migraine headache. Based on these heritable groupings (h(2) = 0.49, 95% CI: 0.41-0.57) individuals were classified as affected (migrainous headache) or unaffected. Genome-wide linkage analysis was performed using genotype data from 105 families with at least 2 affected siblings. In addition to this primary phenotype, linkage analyses were performed for the individual migraine symptoms. Significance levels, corrected for the analysis of multiple traits, were determined empirically via a novel simulation approach. Suggestive linkage for migrainous headache was found on chromosomes 1 (LOD = 1.63; pointwise P = 0.0031), 13 (LOD = 1.63; P = 0.0031), and 20 (LOD = 1.85; P = 0.0018). Interestingly, the chromosome 1 peak was located close to the ATP1A2 gene, associated with familial hemiplegic migraine type 2 (FHM2). Individual symptom analysis produced a LOD score of 1.97 (P = 0.0013) on chromosome 5 (photo/phonophobia), a LOD score of 2.13 (P = 0.0009) on chromosome 10 (moderate/severe pain intensity) and a near significant LOD score of 3.31 (P = 0.00005) on chromosome 13 (pulsating headache). These peaks were all located near regions previously reported in migraine linkage studies. Our results provide important replication and support for the presence of migraine susceptibility genes within these regions, and further support the utility of an LCA-based phenotyping approach and analysis of individual symptoms in migraine genetic research. Additionally, our novel "2-step" analysis and simulation approach provides a powerful means to investigate linkage to individual trait components.

Variants in EMX2 and PTEN do not contribute to risk of endometriosis

Endometriosis has a genetic component, and significant linkage has been found to a region on chromosome 10q. Two candidate genes, EMX2 and PTEN, implicated in both endometriosis and endometrial cancer, lie on chromosome 10q. We hypothesized that variation in EMX2 and/or PTEN could contribute to the risk of endometriosis and may account for some of the linkage signal on 10q. We genotyped single nucleotide polymorphisms (SNPs) in a case-control design to evaluate association between endometriosis and common variations in these two genes. The genotyping and statistical analysis were based on samples collected from Australian volunteers. The cases were 768 unrelated women with surgically confirmed endometriosis selected from affected sister pair (ASP) families participating in the Australian Genes behind Endometriosis Study. The controls were 768 female participants in twin studies who, based on screening questions, did not have a diagnosis of endometriosis. Genotypes of 22 SNPs in the EMX2 gene and 15 SNPs in the PTEN gene were the main outcome measures. Statistical analysis provided measures of linkage disequilibrium and association. Permutation testing showed no globally significant association between any SNPs or haplotypes and endometriosis for either gene. It is unlikely that the EMX2 or PTEN gene variants investigated contribute to risk for initiation and/or development of endometriosis.

KRAS variation and risk of endometriosis

Endometriosis is a common gynaecological disease with symptoms of pelvic pain and infertility which affects 7-10% of women in their reproductive years. Activation of an oncogenic allele of Kirsten rat sarcoma viral oncogene homologue (KRAS) in the reproductive tract of mice resulted in the development of endometriosis. We hypothesized that variation in KRAS may influence risk of endometriosis in humans. Thirty tagSNPs spanning a region of 60.7 kb across the KRAS locus were genotyped using iPLEX chemistry on a MALDI-TOF MassARRAY platform in 959 endometriosis cases and 959 unrelated controls, and data were analysed for association with endometriosis. Genotypes were obtained for most individuals with a mean completion rate of 99.1%. We identified six haplotype blocks across the KRAS locus in our sample. There were no significant differences between cases and controls in the frequencies of individual single-nucleotide polymorphisms (SNPs) or haplotypes. We also developed a rapid method to screen for 11 common KRAS and BRAF mutations on the Sequenom MassARRAY system. The assay detected all mutations previously identified by direct sequencing in a panel of positive controls. No germline variants for KRAS or BRAF were detected. Our results demonstrate that any risk of endometriosis in women because of common variation in KRAS must be very small.

Migraine with aura and migraine without aura are not distinct entities: further evidence from a large Dutch population study

It is often debated whether migraine with aura (MA) and migraine without aura (MO) are etiologically distinct disorders. A previous study using latent class analysis (LCA) in Australian twins showed no evidence for separate subtypes of MO and MA. The aim of the present study was to replicate these results in a population of Dutch twins and their parents, siblings and partners (N = 10,144). Latent class analysis of International Headache Society (IHS)-based migraine symptoms resulted in the identification of 4 classes: a class of unaffected subjects (class 0), a mild form of nonmigrainous headache (class 1), a moderately severe type of migraine (class 2), typically without neurological symptoms or aura (8% reporting aura symptoms), and a severe type of migraine (class 3), typically with neurological symptoms, and aura symptoms in approximately half of the cases. Given the overlap of neurological symptoms and nonmutual exclusivity of aura symptoms, these results do not support the MO and MA subtypes as being etiologically distinct. The heritability in female twins of migraine based on LCA classification was estimated at .50 (95% confidence intervals [CI] .27 - .59), similar to IHS-based migraine diagnosis (h2 = .49, 95% CI .19-.57). However, using a dichotomous classification (affected-unaffected) decreased heritability for the IHS-based classification (h2 = .33, 95% CI .00-.60), but not the LCA-based classification (h2 = .51, 95% CI .23-.61). Importantly, use of the LCA-based classification increased the number of subjects classified as affected. The heritability of the screening question was similar to more detailed LCA and IHS classifications, suggesting that the screening procedure is an important determining factor in genetic studies of migraine.

Genomewide significant linkage to migrainous headache on chromosome 5q21

Familial typical migraine is a common, complex disorder that shows strong familial aggregation. Using latent-class analysis (LCA), we identified subgroups of people with migraine/severe headache in a community sample of 12,245 Australian twins (60% female), drawn from two cohorts of individuals aged 23-90 years who completed an interview based on International Headache Society criteria. We report results from genomewide linkage analyses involving 756 twin families containing a total of 790 independent sib pairs (130 affected concordant, 324 discordant, and 336 unaffected concordant for LCA-derived migraine). Quantitative-trait linkage analysis produced evidence of significant linkage on chromosome 5q21 and suggestive linkage on chromosomes 8, 10, and 13. In addition, we replicated previously reported typical-migraine susceptibility loci on chromosomes 6p12.2-p21.1 and 1q21-q23, the latter being within 3 cM of the rare autosomal dominant familial hemiplegic migraine gene (ATP1A2), a finding which potentially implicates ATP1A2 in familial typical migraine for the first time. Linkage analyses of individual migraine symptoms for our six most interesting chromosomes provide tantalizing hints of the phenotypic and genetic complexity of migraine. Specifically, the chromosome 1 locus is most associated with phonophobia; the chromosome 5 peak is predominantly associated with pulsating headache; the chromosome 6 locus is associated with activity-prohibiting headache and photophobia; the chromosome 8 locus is associated with nausea/vomiting and moderate/severe headache; the chromosome 10 peak is most associated with phonophobia and photophobia; and the chromosome 13 peak is completely due to association with photophobia. These results will prove to be invaluable in the design and analysis of future linkage and linkage disequilibrium studies of migraine.

Genetic and environmental influences on migraine: a twin study across six countries

Migraine is a common neurovascular brain disorder that is manifested in recurrent episodes of disabling headache. The aim of the present study was to compare the prevalence and heritability of migraine across six of the countries that participate in GenomEUtwin project including a total number of 29,717 twin pairs. Migraine was assessed by questionnaires that differed between most countries. It was most prevalent in Danish and Dutch females (32% and 34%, respectively), whereas the lowest prevalence was found in the younger and older Finnish cohorts (13% and 10%, respectively). The estimated genetic variance (heritability) was significant and the same between sexes in all countries. Heritability ranged from 34% to 57%, with lowest estimates in Australia, and highest estimates in the older cohort of Finland, the Netherlands, and Denmark. There was some indication that part of the genetic variance was non-additive, but this was significant in Sweden only. In addition to genetic factors, environmental effects that are non-shared between members of a twin pair contributed to the liability of migraine. After migraine definitions are homogenized among the participating countries, the GenomEUtwin project will provide a powerful resource to identify the genes involved in migraine.

Exclusion of angiotensinogen gene in molecular basis of human hypertension: Sibpair linkage and association analyses in Australian anglo-caucasians

Linkage with essential hypertension has been claimed for a microsatellite marker near the angiotensinogen gene (AGT; chromosome 1q42), as has association for the AGT variants M235T, G(-6)A and A(-20)C. To more rigorously evaluate AGT as a candidate gene for hypertension we performed sibpair analysis with multiple microsatellite markers surrounding this locus and using more sophisticated analysis programs. We also performed an association study of the AGT variants in unrelated subjects with a strong family history (two affected parents). For the linkage study, single and multiplex polymerase chain reaction (PCRs) and automated genescan analysis were conducted on DNA from 175 Australian Anglo-Celtic Caucasian hypertensives for the following markers: D1S2880-(2.1 cM)-D1S213-(2.8 cM)-D1S251-(6.5 cM)-AGT-(2.0 cM) -D1S235. Statistical evaluation of genotype data by nonparametric methods resulted in the following scores: Single-point analysis - SPLINK, P > 0.18; APM method, P > 0.25; ASPEX, MLOD < 0.28; SIB-PAIR, P > 0. 24; Multipoint analysis - MAPMAKER/SIBS, MLOD < 0.24; GENEHUNTER, P > 0.35. Exclusion scores of Lod -4.1 to -5.1 were obtained for these markers using MAPMAKER/SIBS for a lambda(s) of 1.6. The association study of G(-6)A, A(-20)C and M235T variants in 111 hypertensives with strong family history and 190 normotensives with no family history showed significant linkage disequilibrium between particular haplotypes, but we could find no association with hypertension. The present study therefore excludes AGT in the etiology of hypertension, at least in the population of Australian Anglo-Celtic Caucasians studied.

G-protein beta3 subunit gene (GNB3) variant in causation of essential hypertension

-Essential hypertensives display enhanced signal transduction through pertussis toxin-sensitive G proteins. The T allele of a C825T variant in exon 10 of the G protein beta3 subunit gene (GNB3) induces formation of a splice variant (Gbeta3-s) with enhanced activity. The T allele of GNB3 was shown recently to be associated with hypertension in unselected German patients (frequency=0.31 versus 0.25 in control). To confirm and extend this finding in a different setting, we performed an association study in Australian white hypertensives. This involved an extensively examined cohort of 110 hypertensives, each of whom were the offspring of 2 hypertensive parents, and 189 normotensives whose parents were both normotensive beyond age 50 years. Genotyping was performed by polymerase chain reaction and digestion with BseDI, which either cut (C allele) or did not cut (T allele) the 268-bp polymerase chain reaction product. T allele frequency in the hypertensive group was 0.43 compared with 0.25 in the normotensive group (chi2=22; P=0.00002; odds ratio=2.3; 95% CI=1.7 to 3.3). The T allele tracked with higher pretreatment blood pressure: diastolic=105+/-7, 109+/-16, and 128+/-28 mm Hg (mean+/-SD) for CC, CT, and TT, respectively (P=0.001 by 1-way ANOVA). Blood pressures were higher in female hypertensives with a T allele (P=0.006 for systolic and 0.0003 for diastolic by ANOVA) than they were in male hypertensives. In conclusion, the present study of a group with strong family history supports a role for a genetically determined, physiologically active splice variant of the G protein beta3 subunit gene in the causation of essential hypertension.

A comparison of self-nominated and actual speeds in work zones

Despite significant research on drivers’ speeding behavior in work zones, little is known about how well drivers’ judgments of appropriate speeds match their actual speeds and what factors influence their judgments. This study aims to fill these two important gaps in the literature by comparing observed speeds in two work zones with drivers’ self-nominated speeds for the same work zones. In an online survey, drivers nominated speeds for the two work zones based on photographs in which the actual posted speed limits were not revealed. A simultaneous equation modelling approach was employed to examine the effects of driver characteristics on their self-nominated speeds. The results showed that survey participants nominated lower speeds (corresponding to higher compliance rates) than those which were observed. Higher speeds were nominated by males than females, young and middle aged drivers than older drivers, and drivers with truck driving experience than those who drive only cars. Larger differences between nominated and observed speeds were found among car drivers than truck drivers. These differences suggest that self-nominated speeds might not be valid indicators of the observed work zone speeds and therefore should not be used as an alternative to observed speed data.

Eldercare demands, strain, and work engagement: The moderating role of perceived organizational support

Demographic changes give rise to an increasing number of middle-aged employees providing home-based care to an elderly family member. However, the potentially important role of employees' perceptions of organizational support for eldercare has so far not been investigated. The goal of this study was to examine a stressor–strain–outcome model (Koeske & Koeske, 1993) of eldercare strain as a mediator of the relationship between eldercare demands and caregivers' work engagement. Perceived organizational eldercare support was expected to attenuate the positive relationship between eldercare demands and eldercare strain and to buffer the negative relationship between eldercare strain and work engagement. Results of mediation and moderated mediation analyses with data collected from 147 employees providing eldercare supported the hypotheses. The findings suggest that perceived organizational eldercare support is especially beneficial for employees' work engagement when eldercare demands and strain are high.

Distinct subpopulations of gamma delta T cells are present in normal and tumor-bearing human liver

Gamma delta T cells are thought to mediate immune responses at epithelial surfaces. We have quantified and characterized hepatic and peripheral blood gamma delta T cells from 11 normal and 13 unresolved tumor-bearing human liver specimens. gamma delta T cells are enriched in normal liver (6.6% of T cells) relative to matched blood (0.9%; P = 0.008). The majority express CD4(-)CD8(-) phenotypes and many express CD56 and/or CD161. In vitro, hepatic gamma delta T cells can be induced to kill tumor cell lines and release interferon-gamma, tumor necrosis factor-alpha, interleukin-2 and interleukin-4. Analysis of V gamma and V delta chain usage indicated that V delta 3(+) cells are expanded in normal livers (21.2% of gamma delta T cells) compared to blood (0.5%; P = 0.001). Tumor-bearing livers had significant expansions and depletions of gamma delta T cell subsets but normal cytolytic activity. This study identifies novel populations of liver T cells that may play a role in immunity against tumors.

Disease-associated polymorphisms in ERAP1 do not alter endoplasmic reticulum stress in patients with ankylosing spondylitis

The mechanism by which human leukocyte antigen B27 (HLA-B27) contributes to ankylosing spondylitis (AS) remains unclear. Genetic studies demonstrate that association with and interaction between polymorphisms of endoplasmic reticulum aminopeptidase 1 (ERAP1) and HLA-B27 influence the risk of AS. It has been hypothesised that ERAP1-mediated HLA-B27 misfolding increases endoplasmic reticulum (ER) stress, driving an interleukin (IL) 23-dependent, pro-inflammatory immune response. We tested the hypothesis that AS-risk ERAP1 variants increase ER-stress and concomitant pro-inflammatory cytokine production in HLA-B27 + but not HLA-B27-AS patients or controls. Forty-nine AS cases and 22 healthy controls were grouped according to HLA-B27 status and AS-associated ERAP1 rs30187 genotypes: HLA-B27 + ERAP1 risk, HLA-B27 + ERAP1 protective, HLA-B27-ERAP1 risk and HLA-B27-ERAP1 protective. Expression levels of ER-stress markers GRP78 (8 kDa glucose-regulated protein), CHOP (C/EBP-homologous protein) and inflammatory cytokines were determined in peripheral blood mononuclear cell and ileal biopsies. We found no differences in ER-stress gene expression between HLA-B27 + and HLA-B27-cases or healthy controls, or between cases or controls stratified by carriage of ERAP1 risk or protective alleles in the presence or absence of HLA-B27. No differences were observed between expression of IL17A or TNF (tumour necrosis factor) in HLA-B27 + ERAP1 risk, HLA-B27 + ERAP1 protective and HLA-B27-ERAP1 protective cases. These data demonstrate that aberrant ERAP1 activity and HLA-B27 carriage does not alter ER-stress levels in AS, suggesting that ERAP1 and HLA-B27 may influence disease susceptibility through other mechanisms. © 2015 Macmillan Publishers Limited.

Acute fluid shifts influence the assessment of serum vitamin D status in critically ill patients

Introduction Recent reports have highlighted the prevalence of vitamin D deficiency and suggested an association with excess mortality in critically ill patients. Serum vitamin D concentrations in these studies were measured following resuscitation. It is unclear whether aggressive fluid resuscitation independently influences serum vitamin D. Methods Nineteen patients undergoing cardiopulmonary bypass were studied. Serum 25(OH)D3, 1α,25(OH)2D3, parathyroid hormone, C-reactive protein (CRP), and ionised calcium were measured at five defined timepoints: T1 - baseline, T2 - 5 minutes after onset of cardiopulmonary bypass (CPB) (time of maximal fluid effect), T3 - on return to the intensive care unit, T4 - 24 hrs after surgery and T5 - 5 days after surgery. Linear mixed models were used to compare measures at T2-T5 with baseline measures. Results Acute fluid loading resulted in a 35% reduction in 25(OH)D3 (59 ± 16 to 38 ± 14 nmol/L, P < 0.0001) and a 45% reduction in 1α,25(OH)2D3 (99 ± 40 to 54 ± 22 pmol/L P < 0.0001) and i(Ca) (P < 0.01), with elevation in parathyroid hormone (P < 0.0001). Serum 25(OH)D3 returned to baseline only at T5 while 1α,25(OH)2D3 demonstrated an overshoot above baseline at T5 (P < 0.0001). There was a delayed rise in CRP at T4 and T5; this was not associated with a reduction in vitamin D levels at these time points. Conclusions Hemodilution significantly lowers serum 25(OH)D3 and 1α,25(OH)2D3, which may take up to 24 hours to resolve. Moreover, delayed overshoot of 1α,25(OH)2D3 needs consideration. We urge caution in interpreting serum vitamin D in critically ill patients in the context of major resuscitation, and would advocate repeating the measurement once the effects of the resuscitation have abated.

Analysis of body composition in individuals with high bone mass reveals a marked increase in fat mass in women but not men

Context: High bone mass (HBM), detected in 0.2% of dual-energy x-ray absorptiometry (DXA) scans, is characterized by raised body mass index, the basis for which is unclear. Objective: To investigate why body mass index is elevated in individuals with HBM, we characterized body composition and examined whether differences could be explained by bone phenotypes, eg, bone mass and/or bone turnover. Design, Setting, and Participants: We conducted a case-control study of 153 cases with unexplained HBM recruited from 4 UK centers by screening 219 088 DXA scans. Atotal of 138 first-degree relatives (of whom 51 had HBM) and 39 spouses were also recruited. Unaffected individuals served as controls. Main Outcome Measures: We measured fat mass, by DXA, and bone turnover markers. Results: Amongwomen, fat mass was inversely related to age in controls (P<.01), but not in HBM cases (P<.96) in whom mean fat mass was 8.9 [95% CI 4.7, 13.0] kg higher compared with controls (fully adjusted mean difference, P<.001). Increased fat mass in male HBM cases was less marked (gender interaction P = .03). Compared with controls, lean mass was also increased in female HBM cases (by 3.3 [1.2, 5.4] kg; P<.002); however, lean mass increases wereless marked than fat mass increases, resulting in 4.5% lower percentage lean mass in HBM cases (P<.001). Osteocalcin was also lower in female HBM cases compared with controls (by 2.8 [0.1, 5.5]μg/L; P = .04). Differences in fat mass were fully attenuated after hip bone mineral density (BMD) adjustment (P = .52) but unchanged after adjustment for bone turnover (P < .001), whereas the greater hip BMD in female HBM cases was minimally attenuated by fat mass adjustment (P<.001). Conclusions: HBM is characterized by a marked increase in fat mass in females, statistically explained by their greater BMD, but not by markers of bone turnover. Copyright © 2013 by The Endocrine Society.