Within-family outliers: Segregating alleles or environmental effects? A linkage analysis of height from 5815 sibling pairs

Most information in linkage analysis for quantitative traits comes from pairs of relatives that are phenotypically most discordant or concordant. Confounding this, within-family outliers from non-genetic causes may create false positives and negatives. We investigated the influence of within-family outliers empirically, using one of the largest genome-wide linkage scans for height. The subjects were drawn from Australian twin cohorts consisting of 8447 individuals in 2861 families, providing a total of 5815 possible pairs of siblings in sibships. A variance component linkage analysis was performed, either including or excluding the within-family outliers. Using the entire dataset, the largest LOD scores were on chromosome 15q (LOD 2.3) and 11q (1.5). Excluding within-family outliers increased the LOD score for most regions, but the LOD score on chromosome 15 decreased from 2.3 to 1.2, suggesting that the outliers may create false negatives and false positives, although rare alleles of large effect may also be an explanation. Several regions suggestive of linkage to height were found after removing the outliers, including 1q23.1 (2.0), 3q22.1 (1.9) and 5q32 (2.3). We conclude that the investigation of the effect of within-family outliers, which is usually neglected, should be a standard quality control measure in linkage analysis for complex traits and may reduce the noise for the search of common variants of modest effect size as well as help identify rare variants of large effect and clinical significance. We suggest that the effect of within-family outliers deserves further investigation via theoretical and simulation studies.

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.

A case of true hermaphroditism reveals an unusual mechanism of twinning

Traditionally twins are classified as dizygous or fraternal and monozygous or identical (Hall Twinning, 362, 2003 and 735-743). We report a rare case of 46,XX/46,XY twins: Twin A presented with ambiguous genitalia and Twin B was a phenotypically normal male. These twins demonstrate a third, previously unreported mechanism for twinning. The twins underwent initial investigation with 17-hydroxyprogesterone and testosterone levels, pelvic ultrasound and diagnostic laparoscopy. Cytogenetic analysis was performed on peripheral blood cells and skin fibroblasts. Histological examination and Fluorescence in situ hybridization studies on touch imprints were performed on gonadal biopsies. DNA analysis using more than 6,000 DNA markers was performed on skin fibroblast samples from the twins and on peripheral blood samples from both parents. Twin A was determined to be a true hermaphrodite and Twin B an apparently normal male. Both twins had a 46,XX/46,XY chromosome complement in peripheral lymphocytes, skin fibroblasts, and gonadal biopsies. The proportion of XX to XY cells varied between the twins and the tissues evaluated. Most significantly the twins shared 100% of maternal alleles and approximately 50% of paternal alleles in DNA analysis of skin fibroblasts. The twins are chimeric and share a single genetic contribution from their mother but have two genetic contributions from their father thus supporting the existence of a third, previously unreported type of twinning.

Personality, health and lifestyle in a questionnaire family study: A comparison between highly cooperative and less cooperative families

The effect of nonresponse on health and lifestyle measures has received extensive study, showing at most relatively modest effects. Nonresponse bias with respect to personality has been less thoroughly investigated. The present study uses data from responding individuals as a proxy for the missing data of their nonresponding family members to examine the presence of nonresponse bias for personality traits and disorders as well as health and lifestyle traits. We looked at the Big Five personality traits, borderline personality disorder (BPD) features, attention-deficit/hyperactivity disorder, Anger, and several measures of health (Body Mass Index, migraine) and lifestyle (smoking, alcohol use). In general, outcomes tend to be slightly more favorable for individuals from highly cooperative families compared to individuals from less cooperative families. The only significant difference was found for BPD features (p = .001). However, the absolute difference in mean scores is very small, less than 1 point for a scale ranging from 0 to 72. In conclusion, survey data on personality, health and lifestyle are relatively unbiased with respect to nonresponse.

On the probability of dizygotic twins being concordant for two alleles at multiple polymorphic loci

Accurate determination of same-sex twin zygosity is important for medical, scientific and personal reasons. Determination may be based upon questionnaire data, blood group, enzyme isoforms and fetal membrane examination, but assignment of zygosity must ultimately be confirmed by genotypic data. Here methods are reviewed for calculating average probabilities of correctly concluding a twin pair is monozygotic, given they share the same genotypes across all loci for commonly utilized multiplex short tandem repeat (STR) kits.

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.

Association study of the dystrobrevin-binding gene with schizophrenia in Australian and Indian samples

Numerous studies have reported association between variants in the dystrobrevin binding protein 1 (dysbindin) gene (DTNBP1) and schizophrenia. However, the pattern of results is complex and to date, no specific risk marker or haplotype has been consistently identified. The number of single nucleotide polymorphisms (SNPs) tested in these studies has ranged from 5 to 20. We attempted to replicate previous findings by testing 16 SNPs in samples of 41 Australian pedigrees, 194 Australian cases and 180 controls, and 197 Indian pedigrees. No globally significant evidence for association was observed in any sample, despite power calculations indicating sufficient power to replicate several previous findings. Possible explanations for our results include sample differences in background linkage disequilibrium and/or risk allele effect size, the presence of multiple risk alleles upon different haplotypes, or the presence of a single risk allele upon multiple haplotypes. Some previous associations may also represent false positives. Examination of Caucasian HapMap phase II genotype data spanning the DTNBP1 region indicates upwards of 40 SNPs are required to satisfactorily assess all nonredundant variation within DTNBP1 and its potential regulatory regions for association with schizophrenia. More comprehensive studies in multiple samples will be required to determine whether specific DTNBP1 variants function as risk factors for schizophrenia.

A comparison of DNA pools constructed following whole genome amplification for two-stage SNP genotyping designs

Genotyping in DNA pools reduces the cost and the time required to complete large genotyping projects. The aim of the present study was to evaluate pooling as part of a strategy for fine mapping in regions of significant linkage. Thirty-nine single nucleotide polymorphisms (SNPs) were analyzed in two genomic DNA pools of 384 individuals each and results compared with data after typing all individuals used in the pools. There were no significant differences using data from either 2 or 8 heterozygous individuals to correct frequency estimates for unequal allelic amplification. After correction, the mean difference between estimates from the genomic pool and individual allele frequencies was .033. A major limitation of the use of DNA pools is the time and effort required to carefully adjust the concentration of each individual DNA sample before mixing aliquots. Pools were also constructed by combining DNA after Multiple Displacement Amplification (MDA). The MDA pools gave similar results to pools constructed after careful DNA quantitation (mean difference from individual genotyping .040) and MDA provides a rapid method to generate pools suitable for some applications. Pools provide a rapid and cost-effective screen to eliminate SNPs that are not polymorphic in a test population and can detect minor allele frequencies as low as 1% in the pooled samples. With current levels of accuracy, pooling is best suited to an initial screen in the SNP validation process that can provide high-throughput comparisons between cases and controls to prioritize SNPs for subsequent individual genotyping.

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.

Zygosity diagnosis in the absence of genotypic data: An approach using latent class analysis

For zygosity diagnosis in the absence of genotypic data, or in the recruitment phase of a twin study where only single twins from same-sex pairs are being screened, or to provide a test for sample duplication leading to the false identification of a dizygotic pair as monozygotic, the appropriate analysis of respondents' answers to questions about zygosity is critical. Using data from a young adult Australian twin cohort (N = 2094 complete pairs and 519 singleton twins from same-sex pairs with complete responses to all zygosity items), we show that application of latent class analysis (LCA), fitting a 2-class model, yields results that show good concordance with traditional methods of zygosity diagnosis, but with certain important advantages. These include the ability, in many cases, to assign zygosity with specified probability on the basis of responses of a single informant (advantageous when one zygosity type is being oversampled); and the ability to quantify the probability of misassignment of zygosity, allowing prioritization of cases for genotyping as well as identification of cases of probable laboratory error. Out of 242 twins (from 121 like-sex pairs) where genotypic data were available for zygosity confirmation, only a single case was identified of incorrect zygosity assignment by the latent class algorithm. Zygosity assignment for that single case was identified by the LCA as uncertain (probability of being a monozygotic twin only 76%), and the co-twin's responses clearly identified the pair as dizygotic (probability of being dizygotic 100%). In the absence of genotypic data, or as a safeguard against sample duplication, application of LCA for zygosity assignment or confirmation is strongly recommended.

Populational reasoning

This section focuses on systems of reasoning that imagine youth as a unified whole, one that can be researched, talked about, planned for, and managed. Even research that focuses on individuals or specific contexts depends on and reproduces ideas of youth as an identifiable population. This section interrogates the rules and scaffolding of discourses that construct the social spaces in which we problematize and study youth in society. This introduction will set the agenda by addressing four elements of this process: the first addresses the rise of some of the crucial elements of contemporary governance, the instrument and practices through which the notion of the population was able to take shape. The second examines the rise of the personage of “the child,” and how new forms of governance not only utilized this new identity for the purposes of ongoing social management, but also organized its differentiation into a growing array of new social and administrative categories. The third specifically addresses “youth,” examining its various predecessors as targets for moral concern, as well as some of the recent cultural triggers for its formation. Finally, there is an assessment of the contemporary governance of populations of youth, based as it is around its twin existence as a governmental object, a target for an almost endless array of social, educational, legal, and psychological concerns and interventions, but also as an identity, a set of practices of the self.

Does the addition of Clonidine to lumbar nerve root blocks improve outcome? A randomised, prospective, single-blinded controlled pilot study

Purpose To evaluate if adding clonidine to a standard nerve root block containing local anaesthetic and steroid improved the outcome of patients with severe lumbar nerve root pain secondary to MRI proven lumbar disc prolapse. Methods We undertook a single blind, prospective, randomised controlled trial evaluating 100 consecutive patients with nerve root pain secondary to lumbar disc prolapse undergoing trans-foraminal epidural steroid injection either with or without the addition of clonidine. 50 patients were allocated to each arm of the study. The primary outcome measure was the avoidance of a second procedure- repeat injection or micro-discectomy surgery. Secondary outcome measures were also studied: pain scores for leg and back pain using a visual analogue scale (VAS), the Roland Morris Disability Questionnaire (RMDQ) and the Measure Your Own Medical Outcome Profile (MYMOP). Follow up was carried out at 6 weeks, 6 months and 1 year. Results No serious complications occurred. Of the 50 patients who received the addition of clonidine, 56% were classified as successful injections, with no further intervention required, as opposed to 40% who received the standard injection. This difference did not reach statistical significance (p=0.109, chi-squared test). All secondary measures showed no statistically significant differences between the groups except curiously, the standard group who had been classified as successful had better leg pain relief than the clonidine group (p=0.026) at 1 year. Conclusions This pilot study has shown a 16% treatment effect with adding clonidine to lumbar nerve root blocks and that it is a safe injectate for this purpose.

Head motion and Inattention/Hyperactivity share common genetic influences: Implications for fMRI studies of ADHD

Head motion (HM) is a well known confound in analyses of functional MRI (fMRI) data. Neuroimaging researchers therefore typically treat HM as a nuisance covariate in their analyses. Even so, it is possible that HM shares a common genetic influence with the trait of interest. Here we investigate the extent to which this relationship is due to shared genetic factors, using HM extracted from resting-state fMRI and maternal and self report measures of Inattention and Hyperactivity-Impulsivity from the Strengths and Weaknesses of ADHD Symptoms and Normal Behaviour (SWAN) scales. Our sample consisted of healthy young adult twins (N = 627 (63% females) including 95 MZ and 144 DZ twin pairs, mean age 22, who had mother-reported SWAN; N = 725 (58% females) including 101 MZ and 156 DZ pairs, mean age 25, with self reported SWAN). This design enabled us to distinguish genetic from environmental factors in the association between head movement and ADHD scales. HM was moderately correlated with maternal reports of Inattention (r = 0.17, p-value = 7.4E-5) and Hyperactivity-Impulsivity (r = 0.16, p-value = 2.9E-4), and these associations were mainly due to pleiotropic genetic factors with genetic correlations [95% CIs] of rg = 0.24 [0.02, 0.43] and rg = 0.23 [0.07, 0.39]. Correlations between self-reports and HM were not significant, due largely to increased measurement error. These results indicate that treating HM as a nuisance covariate in neuroimaging studies of ADHD will likely reduce power to detect between-group effects, as the implicit assumption of independence between HM and Inattention or Hyperactivity-Impulsivity is not warranted. The implications of this finding are problematic for fMRI studies of ADHD, as failing to apply HM correction is known to increase the likelihood of false positives. We discuss two ways to circumvent this problem: censoring the motion contaminated frames of the RS-fMRI scan or explicitly modeling the relationship between HM and Inattention or Hyperactivity-Impulsivity

Antimicrobial efficacy of a novel silver hydrogel dressing compared to two common silver burn wound dressings: Acticoat™ and PolyMem Silver

A novel burn wound hydrogel dressing has been previously developed which is composed of 2-acrylamido-2-methylpropane sulfonic acid sodium salt with silver nanoparticles. This study compared the antimicrobial efficacy of this novel dressing to two commercially available silver dressings; Acticoat™ and PolyMem Silver(®). Three different antimicrobial tests were used: disc diffusion, broth culture, and the Live/Dead(®) Baclight™ bacterial viability assay. Burn wound pathogens (P. aeruginosa, MSSA, A. baumannii and C. albicans) and antibiotic resistant strains (MRSA and VRE) were tested. All three antimicrobial tests indicated that Acticoat™ was the most effective antimicrobial agent, with inhibition zone lengths of 13.9-18.4mm. It reduced the microbial inocula below the limit of detection (10(2)CFU/ml) and reduced viability by 99% within 4h. PolyMem Silver(®) had no zone of inhibition for most tested micro-organisms, and it also showed poor antimicrobial activity in the broth culture and Live/Dead(®) Baclight™ assays. Alarmingly, it appeared to promote the growth of VRE. The silver hydrogel reduced most of the tested microbial inocula below the detection limit and decreased bacterial viability by 94-99% after 24h exposure. These results support the possibility of using this novel silver hydrogel as a burn wound dressing in the future

Inflammation-driven bone formation in a mouse model of ankylosing spondylitis: Sequential not parallel processes

Background Ankylosing spondylitis (AS) is an immune-mediated arthritis particularly targeting the spine and pelvis and is characterised by inflammation, osteoproliferation and frequently ankylosis. Current treatments that predominately target inflammatory pathways have disappointing efficacy in slowing disease progression. Thus, a better understanding of the causal association and pathological progression from inflammation to bone formation, particularly whether inflammation directly initiates osteoproliferation, is required. Methods The proteoglycan-induced spondylitis (PGISp) mouse model of AS was used to histopathologically map the progressive axial disease events, assess molecular changes during disease progression and define disease progression using unbiased clustering of semi-quantitative histology. PGISp mice were followed over a 24-week time course. Spinal disease was assessed using a novel semi-quantitative histological scoring system that independently evaluated the breadth of pathological features associated with PGISp axial disease, including inflammation, joint destruction and excessive tissue formation (osteoproliferation). Matrix components were identified using immunohistochemistry. Results Disease initiated with inflammation at the periphery of the intervertebral disc (IVD) adjacent to the longitudinal ligament, reminiscent of enthesitis, and was associated with upregulated tumor necrosis factor and metalloproteinases. After a lag phase, established inflammation was temporospatially associated with destruction of IVDs, cartilage and bone. At later time points, advanced disease was characterised by substantially reduced inflammation, excessive tissue formation and ectopic chondrocyte expansion. These distinct features differentiated affected mice into early, intermediate and advanced disease stages. Excessive tissue formation was observed in vertebral joints only if the IVD was destroyed as a consequence of the early inflammation. Ectopic excessive tissue was predominantly chondroidal with chondrocyte-like cells embedded within collagen type II- and X-rich matrix. This corresponded with upregulation of mRNA for cartilage markers Col2a1, sox9 and Comp. Osteophytes, though infrequent, were more prevalent in later disease. Conclusions The inflammation-driven IVD destruction was shown to be a prerequisite for axial disease progression to osteoproliferation in the PGISp mouse. Osteoproliferation led to vertebral body deformity and fusion but was never seen concurrent with persistent inflammation, suggesting a sequential process. The findings support that early intervention with anti-inflammatory therapies will be needed to limit destructive processes and consequently prevent progression of AS.