GenePy - a score for estimating gene pathogenicity in individuals using next-generation sequencing data.

BACKGROUND: Next-generation sequencing is revolutionising diagnosis and treatment of rare diseases, however its application to understanding common disease aetiology is limited. Rare disease applications binarily attribute genetic change(s) at a single locus to a specific phenotype. In common diseases, where multiple genetic variants within and across genes contribute to disease, binary modelling cannot capture the burden of pathogenicity harboured by an individual across a given gene/pathway. We present GenePy, a novel gene-level scoring system for integration and analysis of next-generation sequencing data on a per-individual basis that transforms NGS data interpretation from variant-level to gene-level. This simple and flexible scoring system is intuitive and amenable to integration for machine learning, network and topological approaches, facilitating the investigation of complex phenotypes. RESULTS: Whole-exome sequencing data from 508 individuals were used to generate GenePy scores. For each variant a score is calculated incorporating: i) population allele frequency estimates; ii) individual zygosity, determined through standard variant calling pipelines and; iii) any user defined deleteriousness metric to inform on functional impact. GenePy then combines scores generated for all variants observed into a single gene score for each individual. We generated a matrix of ~ 14,000 GenePy scores for all individuals for each of sixteen popular deleteriousness metrics. All per-gene scores are corrected for gene length. The majority of genes generate GenePy scores < 0.01 although individuals harbouring multiple rare highly deleterious mutations can accumulate extremely high GenePy scores. In the absence of a comparator metric, we examine GenePy performance in discriminating genes known to be associated with three common, complex diseases. A Mann-Whitney U test conducted on GenePy scores for this positive control gene in cases versus controls demonstrates markedly more significant results (p = 1.37 × 10- 4) compared to the most commonly applied association tool that combines common and rare variation (p = 0.003). CONCLUSIONS: Per-gene per-individual GenePy scores are intuitive when assessing genetic variation in individual patients or comparing scores between groups. GenePy outperforms the currently accepted best practice tools for combining common and rare variation. GenePy scores are suitable for downstream data integration with transcriptomic and proteomic data that also report at the gene level.

A critical hybrid realist-outcomes systematic review of relationships between medical education programmes and communities: BEME Guide No. 35.

BACKGROUND: The relationships between medical schools and communities have long inspired and troubled medical education programmes. Successive models of community-oriented, community-based and community-engaged medical education have promised much and delivered to varying degrees. A two-armed realist systematic review was undertaken to explore and synthesize the evidence on medical school-community relationships. METHOD: One arm used standard outcomes criteria (Kirkpatrick levels), the other a realist approach seeking out the underlying contexts, mechanisms and outcomes. 38 reviewers completed 489 realist reviews and 271 outcomes reviews; 334 articles were reviewed in the realist arm and 181 in the outcomes arm. Analyses were based on: descriptive statistics on both articles and reviews; the outcomes involved; the quality of the evidence presented; realist contexts, mechanisms, and outcomes; and an analysis of underlying discursive themes. FINDINGS: The literature on medical school-community relationships is heterogeneous and largely idiographic, with no common standards for what a community is, who represents communities, what a relationship is based on, or whose needs are or should be being addressed or considered. CONCLUSIONS: Community relationships can benefit medical education, even if it is not always clear why or how. There is much opportunity to improve the quality and precision of scholarship in this area.

Variance components analyses of multiple asthma traits in a large sample of Australian families ascertained through a twin proband.

BACKGROUND: Intermediate phenotypes are often measured as a proxy for asthma. It is largely unclear to what extent the same set of environmental or genetic factors regulate these traits. OBJECTIVE: Estimate the environmental and genetic correlations between self-reported and clinical asthma traits. METHODS: A total of 3,073 subjects from 802 families were ascertained through a twin proband. Traits measured included self-reported asthma, airway histamine responsiveness (AHR), skin prick response to common allergens including house dust mite (Dermatophagoides pteronyssinus [D. pter]), baseline lung function, total serum immunoglobulin E (IgE) and eosinophilia. Bivariate and multivariate analyses of eight traits were performed with adjustment for ascertainment and significant covariates. RESULTS: Overall 2,716 participants completed an asthma questionnaire and 2,087 were clinically tested, including 1,289 self-reported asthmatics (92% previously diagnosed by a doctor). Asthma, AHR, markers of allergic sensitization and eosinophilia had significant environmental correlations with each other (range: 0.23-0.89). Baseline forced expiratory volume in 1 s (FEV(1)) showed low environmental correlations with most traits. Fewer genetic correlations were significantly different from zero. Phenotypes with greatest genetic similarity were asthma and atopy (0.46), IgE and eosinophilia (0.44), AHR and D. pter (0.43) and AHR and airway obstruction (-0.43). Traits with greatest genetic dissimilarity were FEV(1) and atopy (0.05), airway obstruction and IgE (0.07) and FEV(1) and D. pter (0.11). CONCLUSION: These results suggest that the same set of environmental factors regulates the variation of many asthma traits. In addition, although most traits are regulated to great extent by specific genetic factors, there is still some degree of genetic overlap that could be exploited by multivariate linkage approaches.

Melanocortin-1 receptor polymorphisms and risk of melanoma: is the association explained solely by pigmentation phenotype?

Risk of cutaneous malignant melanoma (CMM) is increased in sun-exposed whites, particularly those with a pale complexion. This study was designed to investigate the relationship of the melanocortin-1 receptor (MC1R) genotype to CMM risk, controlled for pigmentation phenotype. We report the occurrence of five common MC1R variants in an Australian population-based sample of 460 individuals with familial and sporadic CMM and 399 control individuals-and their relationship to such other risk factors as skin, hair, and eye color; freckling; and nevus count. There was a strong relationship between MC1R variants and hair color and skin type. Moreover, MC1R variants were found in 72% of the individuals with CMM, whereas only 56% of the control individuals carried at least one variant (P<.001), a finding independent of strength of family history of melanoma. Three active alleles (Arg151Cys, Arg160Trp, and Asp294His), previously associated with red hair, doubled CMM risk for each additional allele carried (odds ratio 2.0; 95% confidence interval 1. 6-2.6). No such independent association could be demonstrated with the Val60Leu and Asp84Glu variants. Among pale-skinned individuals alone, this association between CMM and MC1R variants was absent, but it persisted among those reporting a medium or olive/dark complexion. We conclude that the effect that MC1R variant alleles have on CMM is partly mediated via determination of pigmentation phenotype and that these alleles may also negate the protection normally afforded by darker skin coloring in some members of this white population.

Perceptual variation in categorizing individuals according to American College of Rheumatology classification criteria for hand, knee, and hip osteoarthritis (OA): observations based on an Australian Twin Registry study of OA.

OBJECTIVE: The American College of Rheumatology (ACR) classification criteria for osteoarthritis (OA) permit the categorization of individuals for hand, knee, and hip OA and are of defined sensitivity and specificity. They depend on both clinical and radiographic aspects of OA. The clinical diagnosis of OA in the peripheral skeleton is dependent on the skilled examination of several clinical features characteristic of the condition, while the interpretation of radiographs is a perceptual skill based on appreciating specific structural features on plain radiographs. We investigated the interrater reliability of the ACR classification criteria for OA when applied in a community based sample. METHODS: The study was part of a multifaceted diagnostics protocol, evaluating methodologic issues, in the conduct of genetic research in OA. From a cohort of 118 pairs of twins registered with the Australian Twins Registry (ATR), standard clinical examinations of hands, knees, and hips were performed on 74 complete and 11 incomplete pairs of twins over age 50 years. The pairs were selected to represent both twin pairs who had previously self-reported a diagnosis of OA, as well as those who had not. Rheumatologists who performed the assessments were blind to the original self-report. All subjects were examined independently by one of 2 pairs (NB/AK or NB/KM) of consultant rheumatologists, blind to one another's assessments. Each rheumatologist separately assessed the hands, knees, and hips, rating them clinically by ACR criteria for OA. The observations were made without reference to any radiographic or serologic information. RESULTS: Interrater agreement was different for the 3 different anatomic areas and was different for the 2 pairs of rheumatologists. The actual (observed) interrater agreements based on ACR clinical criteria were as follows: hand OA NB/AK = 0.92, NB/KM = 1.00; knee OA NB/AK = 0.94, NB/KM = 0.92; hip OA NB/AK = 0.98, NB/KM = 0.97. Interrater agreement based on ACR clinical criteria, as assessed by the adjusted kappa statistic, was as follows: hand OA NB/AK = 0.84, NB/KM = 1.00; knee OA NB/AK = 0.87, NB/KM = 0.84; hip OA NB/AK = 0.95, NB/KM = 0.93. CONCLUSION: Since clinical agreement was extremely high in all 3 anatomic sites, and for both pairs of assessors, we conclude that for genetic epidemiology purposes, subjects can be examined by a single experienced rheumatologist using the ACR classification criteria.

Perceptual variation in grading hand, hip and knee radiographs: observations based on an Australian twin registry study of osteoarthritis.

OBJECTIVE: The radiographic diagnosis of osteoarthritis (OA) in the peripheral skeleton is dependent on the skilled examination of several morphological characteristics of the condition as visualised on plain radiographs. However, the process is perceptual and generally enhanced by comparison against photographic standards. This study assessed the intra-rater and inter-rater reliability of radiologists experienced in reporting hand, hip and knee films derived from a community-based sample when using the photographic atlas recently developed by Burnett et al. METHODS: This study was part of a multifaceted diagnostics protocol, evaluating methodological issues, in the conduct of genetic research in osteoarthritis. From a cohort of 118 twin pairs, registered with the Australian Twins Registry (ATR), standard clinical examinations were performed on 74 complete and 11 incomplete pairs of twins over age 50 years, followed by standard AP hand, AP pelvis and AP standing radiographs of the knees. The pairs were selected both to represent twin pairs who had previously self reported a diagnosis of OA, as well as those who had not. Radiologists read the films blind to the original self reported diagnosis and without reference to their pairing. The films were read by comparison against photographic standards and were scored according to specific features. All films were read independently by two consultant radiologists blind to one another's assessments, and selected films were thereafter assigned for rereading. Inter-rater and intra-rater agreement were different for different features, different anatomic areas, and, for the former, were different for the two radiologists. RESULTS: Inter-rater agreement was different for different anatomic areas, different radiographic features, and the two radiologists. Intra-rater agreement for the presence or absence of OA was as follows: actual observed agreement = 0.79 to 0.97 and 0.83 to 0.98; adjusted kappa statistic = 0.58 to 0.94 and 0.67 to 0.96; inter-rater agreement was as follows: actual observed agreement = 0. 77 to 0.97; adjusted kappa statistic = 0.54 to 0.94. Agreement was generally high in most of the principal target joints for OA: DIP, PIP, 1st CMC, hip and knee. CONCLUSIONS: Although assessor agreement was not perfect, it is concluded that for genetic epidemiology purposes, while duplicate assessments may be advantageous, it is possible for radiographs to be examined accurately by a single experienced assessor. However, for less experienced assessors independent examinations should be made by at least two assessors and either a consensus reached on disparate examinations or an algorithm developed to adjudicate any discrepancies.

A major quantitative-trait locus for mole density is linked to the familial melanoma gene CDKN2A: a maximum-likelihood combined linkage and association analysis in twins and their sibs.

Important risk factors for melanoma are densely clustered melanocytic nevi (common moles) and mutations in the p16 (CDKN2A) gene. Nevi may be subclassified as raised or flat. In our sample, raised nevi were 27% of the total, and the two kinds had a correlation of.33. Correlations for total-nevus count (TNC) in 153 MZ and 199 DZ twin pairs were.94 and.60, respectively, which are compatible with a very-high degree of genetic determination. We hypothesized that some of the genetic variance might be due to variation in the p16 gene. Analysis of linkage to a highly polymorphic marker (D9S942), located close to p16, detected quantitative-trait-loci (QTL) effects accounting for 27% of variance in TNC, rising to 33% if flat but not raised moles were considered. Total heritability was higher for raised (.69) than for flat (.42) moles, but QTL linkage was 0 for raised moles, whereas it accounted for 80% of the heritability of flat moles; additionally, family environment accounted for only 15% of variance in raised versus 46% in flat moles. These findings suggest that raised and flat nevi have very different etiologies. Longer alleles at D9S942 were associated with higher flat-mole counts, and a novel modification to a within-sibship association test showed that this association is genuine and not due to population stratification, although it accounts for only 1% of total variance. Since germline mutations in the exons of CDKN2A are rare, it is likely that variants in the noncoding regions of this gene, or in another gene nearby, are responsible for this major determinant of moliness and, hence, of melanoma risk.

Differential clinical and motor control function in a pair of monozygotic twins with Huntington's disease.

We report a pair of monozygotic Huntington's disease (HD) twins who, although sharing identical CAG repeat lengths, not only present with marked differences in clinical symptoms but also behavioral abilities as measured by our experimental procedures. Both HD twins and two healthy control subjects were tested twice over 2 years. Patient A was generally more impaired at a motor level, whereas Patient B showed greater attentional impairment; Patient B, however, showed more progressive deterioration. The control subjects' performance remained consistent over the 2-year interval. Patient A clinically had the more hyperkinetic hypotonic variant of the disease, whereas Patient B, who was the more impaired, presented with a more hypokinetic hypertonic (rigid) variant. The influences of epigenetic pre- and postnatal environmental factors should not be ignored.

Cognitive performance at school age of very low birth weight infants with bronchopulmonary dysplasia.

The hypothesis was that bronchopulmonary dysplasia (BPD) adversely affects cognitive performance at school age. This prospective cohort study examined three groups of children at 8 to 10 years of age. Group 1 (n = 311) consisted of very low birth weight (VLBW) infants without BPD and Group 2 (n = 95) consisted of VLBW infants with BPD. Group 3 (n = 188) consisted of term infants without BPD. Regression analysis determined the effect of BPD on eight performance measures while controlling for possible confounding variables. Children in Group 3 had the best performance and children in Group 2 had the poorest performance on all measures. These differences were significant (p = .0001) for the Full Scale IQ, Performance IQ, and reading and math grades and ages. Children in Groups 3 and 1 performed better than children in Group 2 for the Verbal IQ (p = .0001) and the Developmental Test of Visual-Motor Integration (p = .0012), but for these two measures there was no significant difference between children in Groups 3 and 1. Thus, poorer performance was found in VLBW infants with a history of BPD.

Perceptual variation in categorizing individual peripheral joints for the presence or absence of osteoarthritis using a standard homunculus: observations based on an australian twin registry study of osteoarthritis.

The clinical diagnosis of osteoarthritis in the peripheral skeleton is dependent on the skilled examination of several features characteristic of the condition. However, we have previously observed that even highly experienced rheumatologists in Australia and Canada may not completely agree on the clinical examination in individuals with osteoarthritis (OA), rheumatoid arthritis, ankylosing spondylitis, fibromyalgia, scleroderma and painful shoulder.

Complete sequence and polymorphism study of the human TYRP1 gene encoding tyrosinase-related protein 1.

The complete 24,667 nucleotide sequence spanning the human TYRP1 gene has been determined from the inserts of two overlapping lambda clones. A LINE-1 repeat element is immediately adjacent to and may demarcate the immediate 5' promoter region of the gene. A search for polymorphism within the seven TYRP1 coding exons has been performed by an RNase mismatch detection procedure. Analysis of the TYRP1 gene in 100 Caucasian individuals of varying hair color has found no amino acid sequence variation nor revealed any hemizygous mutant allele in the hypopigmented phenotype of two 9p- syndrome patients.

Characterization of melanocyte stimulating hormone receptor variant alleles in twins with red hair.

The association between MSHR coding region variation and hair colour in humans has been examined by genotyping 25 red haired and 62 non-red Caucasians, all of whom were 12 years of age and members of a twin pair study. Twelve amino acid substitutions were seen at 11 different sites, nine of these being newly described MSHR variants. The previously reported Val92Met allele shows no association with hair colour, but the three alleles Arg151Cys, Arg160Trp and Asp294His were associated with red hair and one Val60Leu variant was most frequent in fair/blonde and light brown hair colours. Variant MSHR genotypes are associated with lighter skin types and red hair (P < 0.001). However, comparison of the MSHR genotypes in dizygotic twin pairs discordant for red hair colour indicates that the MSHR gene cannot be solely responsible for the red hair phenotype, since five of 13 pairs tested had both haplotypes identical by state (with three of the five having both identical by descent). Rather, it is likely that additional modifier genes exist, making variance in the MSHR gene necessary but not always sufficient, for red hair production.

Transfer of attaching and effacing from a strain of enteropathogenic Escherichia coli to E. coli K-12.

The ability to cause attaching and effacing (AE) lesions in intestinal epithelial cells is an essential virulence trait of enteropathogenic E. coli (EPEC) that requires several chromosomal genes acting in concert with one another. In this study, we show that the ability to cause AE lesions can be transferred by conjugal mating from a high frequency recombinant (Hfr) derivative of a rabbit EPEC strain, E. coli RDEC-1, to a strain of E. coli K-12. Although the recipient acquired a considerable amount of donor DNA during the transfer process, it expressed the AE phenotype phenotype only weakly. The findings suggest the AE is a multigene phenomenon, the genes for which may not reside on a single region of the bacterial chromosome.

Adherence characteristics of attaching and effacing strains of Escherichia coli from rabbits.

Twelve strains of Escherichia coli previously reported to cause diarrhea in rabbits were examined for properties associated with virulence. Ten strains met the criteria for classification as enteropathogenic E. coli in that they were diarrheagenic strains that evoked attaching-effacing lesions in the small intestine and did not produce detectable enterotoxins or cytotoxins. These bacteria exhibited a variety of patterns when investigated for adherence to HEp-2 epithelial cells. Although several strains displayed localized and/or diffuse adherence to epithelial cells, they did not hybridize with DNA probes that recognize the genes responsible for these phenotypes in diarrheagenic E. coli from humans. The bacteria also varied in their ability to bind to erythrocytes and intestinal brush borders from various animal species. Six strains adhered to rabbit brush borders; two of these also adhered to brush borders from other animals. Two strains that did not adhere to rabbit brush borders adhered to those from guinea pigs or sheep. Only one of the strains investigated carried AF/R1 fimbriae, which are believed to govern the host specificity of this category of diarrheagenic E. coli. This strain was E. coli RDEC-1, which remains the only E. coli strain to date that is known to carry fimbriae of this type. The results indicate that although diarrheagenic E. coli strains from rabbits may have common properties associated with the ability to produce attaching-effacing lesions, they differ from each other and from enteropathogenic E. coli of humans in terms of some of the adhesins that mediate binding to eukaryotic cells.

Examination of archetypal strains of enteropathogenic Escherichia coli for properties associated with bacterial virulence.

Nine strains of Escherichia coli isolated from infants with diarrhoea between 1947 and 1960 and designated "enteropathogenic" were examined for phenotypic and genetic characters associated with virulence. Each strain belonged to a different serotype. All the isolates were historically significant in that they were amongst the first strains of E. coli reported to be causally associated with infantile diarrhoea. Five strains possessed the virulence properties of class I enteropathogenic E. coli (EPEC). All these strains were isolated originally from symptomatic children during outbreaks of diarrhoea. Two isolates from sporadic cases of diarrhoea fulfilled the criteria for classification as class II EPEC. One strain was identified as enteroaggregative E. coli and the other carried no known virulence-associated properties. These findings indicate that most early isolates of E. coli which were designated "enteropathogenic" were indeed EPEC, as currently defined.

Morphology and local structure in labyrinthine stripe domain phase.

Analysis of globally disordered, nonequilibrium "labyrinthine" stripe-domain patterns in thin ferrimagnetic garnet films has revealed a well-defined local structure containing an oblong polygonal plaquette as the fundamental motif. Two types of labyrinths were found: one having topological defects, the other composed of a single, unbranched, meandering line. These patterns emerge when the labyrinthine state is approached either by heating at constant magnetic field or by demagnetizing from saturation at constant temperature. Size and aspect ratios of the oblong polygonal plaquettes prove to be independent of the choice of these two mutually orthogonal trajectories within the phase diagram, which is surprising in view of the different mechanisms and concomitant topological constraints governing the evolution of disorder. The significance of this unique local structure is discussed in the general context of defectmediated melting of two-dimensional stripe phases.

Regional blood-to-tissue transport in an irradiated rat glioma model.

To assess vascular permeability in intracerebral grafts of the 36B-10, F-344 rat glioma following 20 Gy 137Cs whole brain irradiation, the blood-to-tissue transport constant, K, of [14C]-alpha-aminoisobutyric acid (AIB) was measured with quantitative autoradiography. Mean, 90th percentile, and 95th percentile values of K were determined in individual tumors and in treatment groups. In 15-day-old unirradiated control tumors, mean, 90th percentile, and 95th percentile values of K were, respectively, 11.3, 18.4, and 20.8 ml kg-1 min-1. In 15-day-old tumors irradiated on Day 14 (Day 1 postirradiation tumors) the K values were 5.9, 9.4, and 10.4, all of which were significantly less than the respective control values (P less than 0.01). In 16-day-old tumors irradiated on Day 14 (Day 2 postirradiation tumors), the K values were 10.8, 15.0, and 16.0, respectively, none of which was significantly different from control tumors. Mean K values for Day 2 vs Day 1 postirradiation tumors (10.8 vs 5.9) yielded P less than 0.05, but the 90th percentile and 95th percentile values for Day 2 vs Day 1 yielded 0.05 less than P less than 0.10. Separate experiments measured AIB and 86RbCl uptake in 36B-10 cells in vitro 1 and 2 days following 20 Gy irradiation to assess whether this radiation dose reduced the capacity of tumor cells to trap AIB or Rb+. Irradiation did not reduce the accumulation of either tracer, but rather was associated with an increased accumulation of AIB. Therefore, the AIB transport data suggest that vascular permeability and/or surface area decreases significantly in the day following 20 Gy irradiation and that this decrease reverses by the second day following irradiation.

Blood flow in an experimental rat brain tumor by tissue equilibration and indicator fractionation.

The tissue equilibration technique (Kety) was compared with the indicator fractionation technique for the measurement of blood flow to normal brain and an experimental brain tumor in the rat. The tumor was a cloned astrocytic glioma implanted in the cerebral hemisphere of F-344 rats. I-125 Iodoantipyrine, using a rising infusion for one minute, was used for the tissue equilibration technique. C-14 butanol, injected as a bolus 8 seconds before sacrifice, was used for the indicator fractionation technique. Samples were assayed using liquid scintillation counting and the iodoantipyrine results were regressed against the butanol results. For normal tissue R = 0.832, SEE = 0.115 ml/g/min, and Slope = 0.626. For tumor R = 0.796, SEE = 0.070 ml/g/min, and Slope = 0.441. The iodoantipyrine tissue/blood partition coefficient for normal hemisphere (gray and white matter) was 0.861 +/-0.037 (SD) and for tumor was 0.876 +/-0.042. The indicator fractionation technique with C-14 butanol underestimated blood flow in a consistent manner, probably because of incomplete extraction, early washout of activity from tissue and from evaporation of butanol during processing. Our experiments revealed no differences between tumor and normal brain tissue that might invalidate the comparison of iodoantipyrine blood flow results in brain tumors and surrounding normal brain.

The wedge filter technique for convex boundary estimation.

This paper describes a method for segmentation of convex shaped image regions. The wedge filter technique first employs the converging squares algorithm [1] to locate a region of interest. Then a region oriented boundary estimation technique, called the wedge filter, is applied. This wedge filter entails angular filtering and subsampling, and boundary interpolation. The technique is more capable of segmenting noncircular shapes than some earlier methods based on the Hough transform. In addition, unlike many edge-based segmentation schemes, this method is relatively tolerant to edge gaps and to blurred or thick edges. This technique is tested on a number of synthesized images over a range of convex shapes, for different algorithm parameters, and under various conditions of region size and image noise. In addition, the technique has been applied to segmentation of liver cell nuclei in light microscope images of human liver tissue.