exomeSuite: Whole exome sequence variant filtering tool for rapid identification of putative disease causing SNVs/indels.

Exome and whole-genome analyses powered by next-generation sequencing (NGS) have become invaluable tools in identifying causal mutations responsible for Mendelian disorders. Given that individual exomes contain several thousand single nucleotide variants and insertions/deletions, it remains a challenge to analyze large numbers of variants from multiple exomes to identify causal alleles associated with inherited conditions. To this end, we have developed user-friendly software that analyzes variant calls from multiple individuals to facilitate identification of causal mutations. The software, termed exomeSuite, filters for putative causative variants of monogenic diseases inherited in one of three forms: dominant, recessive caused by a homozygous variant, or recessive caused by two compound heterozygous variants. In addition, exomeSuite can perform homozygosity mapping and analyze the variant data of multiple unrelated individuals. Here we demonstrate that filtering of variants with exomeSuite reduces datasets to a fraction of a percent of their original size. To the best of our knowledge this is the first freely available software developed to analyze variant data from multiple individuals that rapidly assimilates and filters large data sets based on pattern of inheritance.

Uptake of abdominal aortic aneurysm screening. A cohort study.

BACKGROUND: Abdominal aortic aneurysms (AAA) are responsible for 1.4% of UK deaths. Deprivation is a risk factor for AAA. Screening reduces AAA related mortality and is cost effective if uptake remains high. The Highland aneurysm screening programme (HASP) began in 2001 offering screening to men in a sparsely populated area. The aim was to identify whether uptake varies with deprivation or rurality, in the context of an established programme. METHODS: Retrospective interrogation of HASP records was performed on all men offered screening from 2001 until 2010. Deprivation and rurality status were derived from postcode of residence (SIMD'09 and URC'08) and the relationships with screening uptake were examined. RESULTS: Mean uptake over the decade was 90.1%. There was a strong association between deprivation and uptake, which ranged from 79.5% in the most deprived population to 97.5% in the least deprived (p < 0.001). The odds of men who were least deprived attending was 10.6 times higher than those who were most deprived (p < 0.001). Higher uptake was observed in more rural areas (p = 0.02). When combined in a logistic regression model, only deprivation remained significant, indicating any apparent effect of rurality was explained by deprivation. No change was observed in the mean aortic diameter of 65-year-old men or the incidence of AAA. CONCLUSION: HASP has a high uptake even in the most deprived and rural populations, demonstrating that programme design has overcome any potential rural disadvantage. A gradient of uptake associated with deprivation remains, although even the most deprived have an uptake of almost 80%.

Practical aspects of equine parasite control: a review based upon a workshop discussion consensus.

Development of resistance of several important equine parasites to most of the available anthelmintic drug classes has led to a reconsideration of parasite control strategies in many equine establishments. Routine prophylactic treatments based on simple calendar-based schemes are no longer reliable and veterinary equine clinicians are increasingly seeking advice and guidance on more sustainable approaches to equine parasite control. Most techniques for the detection of equine helminth parasites are based on faecal analysis and very few tests have been developed as diagnostic tests for resistance. Recently, some molecular and in vitro based diagnostic assays have been developed and have shown promise, but none of these are currently available for veterinary practice. Presently, the only reliable method for the detection of anthelmintic resistance is a simple faecal egg count reduction test, and clinicians are urged to perform such tests on a regular basis. The key to managing anthelmintic resistance is maintaining parasite refugia and this concept is discussed in relation to treatment strategies, drug rotations and pasture management. It is concluded that treatment strategies need to change and more reliance should now be placed on surveillance of parasite burdens and regular drug efficacy tests are also recommended to ensure continuing drug efficacy. The present review is based upon discussions held at an equine parasite workshop arranged by the French Equine Veterinary Association (Association Vétérinaire Equine Française, AVEF) in Reims, France, in October 2008.

Advances in imaging of Stargardt disease.

Stargardt disease (STGD1) is an autosomal-recessively inherited condition often associated with mutations in ABCA4 and characterized by accumulation of autofluorescent lipofuscin deposits in the retinal pigment epithelium (RPE). Non-invasive imaging techniques including fundus autofluorescence (FAF), spectral domain optical coherence tomography (SD-OCT) and adaptive optics scanning laser ophthalmoscopy (AOSLO) have the potential to improve understanding of vision loss in patients with STGD. We describe a comprehensive approach to the study of patients with STGD. Measures of retinal structure and FAF were correlated with visual function including best-corrected visual acuity (BCVA), color vision, kinetic and static perimetry, fundus-guided microperimetry and full-field and multifocal electroretinography. Mutation analysis of the ABCA4 gene was carried out by sequencing the complete coding region. Preliminary data suggest that a combination of imaging modalities may provide a sensitive measure of disease progression and response to experimental therapies in patients with STGD.

Expression of reverse cholesterol transport proteins ATP-binding cassette A1 (ABCA1) and scavenger receptor BI (SR-BI) in the retina and retinal pigment epithelium.

AIMS: Excessive lipid accumulation in Bruch's membrane (BrM) is a hallmark of ageing, the major risk factor for age-related macular degeneration (AMD). Retinal pigment epithelial (RPE) cells may utilise reverse cholesterol transport (RCT) activity to move lipid into BrM, mediated through ATP-binding cassette A1 (ABCA1) and scavenger receptor BI (SR-BI). METHODS: ABCA1 expression was assessed by reverse transcription polymerase chain reaction (RT-PCR) and western blotting of human RPE cell extracts. Lipid transport assays were performed using radiolabelled photoreceptor outer segments (POS). ABCA1 and SR-BI expression was examined in normal mouse eyes by immunofluorescence staining. BrMs of ABCA1 and SR-BI heterozygous mice were examined microscopically. RESULTS: Human RPE cells expressed ABCA1 mRNA and protein. The ABCA1 and SR-BI inhibitor glyburide (also known as glibenclamide) abolished basal transport of POS-derived lipids in RPE cells in the presence of high-density lipoprotein. Mouse retina and RPE expressed ABCA1 and SR-BI. SR-BI was highly expressed in RPE. BrMs were significantly thickened in SR-BI heterozygous mice, but not in ABCA1 heterozygous mice. CONCLUSION: RPE cells express ABCA1 and SR-BI. This implies a significant role for SR-BI and ABCA1 in lipid transport and RCT in the retina and RPE.

The successful use of recombinant factor VIIa in a patient with vascular-type Ehlers-Danlos syndrome.

Vascular-type Ehlers-Danlos syndrome is an inherited connective tissue disorder resulting in an increased risk of serious peri-operative bleeding during surgery for spontaneous organ or vessel rupture. The excessive bleeding may result in coagulopathy, and thus compound the difficulty in securing surgical haemostasis. With the advent of recombinant factor VIIa, a new therapy has become available for the management of intractable surgical bleeding.

Unusual hindgut malrotation and associated mesenteric vascular abnormality presenting as large bowel obstruction in an adult.

We report an unusual and not previously described congenital hindgut malrotation presenting as large bowel obstruction in an adult.

Factors affecting attrition in a longitudinal study of patients with AIDS.

Anecdotal data have suggested that retention of HIV-infected patients with immune recovery in longitudinal studies may be difficult as they resume normal activities. This study evaluated risk factors for attrition among patients with AIDS in a cohort study in the era of highly active antiretroviral therapy. Patients with AIDS enrolled in the Longitudinal Study of Ocular Complications of AIDS were evaluated every three months with demographic, clinical and laboratory data collected. Lost to follow-up was defined as any patient who missed all study visits and could not be contacted for 12 consecutive months, who had not died and who did not re-enter the study at a later date. Of the 1,052 patients studied, 77 (7.3%) were lost to follow-up (rate = 0.03/person year). In the multivariate analysis, factors associated with attrition were CD4+ T-cell count category (hazard ratio (HR) = 2.03; 95%CI: 1.01, 4.24; P = 0.05 for CD4+ count < or = 50 cells/microL and HR = 1.96; 95%CI: 1.12, 3.40; P = 0.02 for CD4+ count 51-200 cells/microL) and detectable HIV viral load (HR = 1.29; 95%CI: 1.07, 1.53; P < 0.001 for HIV viral load >400 copies/mL). These data suggest that patients with compromised immunologic status are at an increased risk for being lost to follow-up.

Light and inherited retinal degeneration.

Light deprivation has long been considered a potential treatment for patients with inherited retinal degenerative diseases, but no therapeutic benefit has been demonstrated to date. In the few clinical studies that have addressed this issue, the underlying mutations were unknown. Our rapidly expanding knowledge of the genes and mechanisms involved in retinal degeneration have made it possible to reconsider the potential value of light restriction in specific genetic contexts. This review summarises the clinical evidence for a modifying role of light exposure in retinal degeneration and experimental evidence from animal models, focusing on retinitis pigmentosa with regional degeneration, Oguchi disease, and Stargardt macular dystrophy. These cases illustrate distinct pathophysiological roles for light, and suggest that light restriction may benefit carefully defined subsets of patients.

Children's surgery performed by adult general surgeons in Scotland: the present and future.

OBJECTIVE: The aim of this study was to determine the extent and the future of paediatric surgery performed in Scotland outside of the designated surgical paediatric centres. MATERIALS AND METHODS: An anonymous questionnaire was sent to all 111 Scottish members of the Association of Surgeons of Great Britain and Ireland. There was a response rate of 69%. RESULTS: Overall, 45% of responders operated on children. This was independent of the surgeon's age but was related to the type of hospital that the surgeon worked in. Eighty-four per cent of responders had a lower age limit under which they would not operate and 94% stated that there were specific circumstances where they would not operate. A mean of 18.5 elective procedures (range 0-250, median two) and six emergency procedures (range 0-30, median five) were carried out by each surgeon operating on children under the age of five per annum. Only 13% of responders thought that their successor would operate on children. CONCLUSIONS: Non-specialist paediatric surgery in Scotland is currently provided by a significant number of surgeons whose successors will not continue to provide a comparative paediatric service. This has implications for local provision of care, emergency management and capacity of existing children's hospitals in the future.

Screening for abdominal aortic aneurysm in a geographically isolated area.

BACKGROUND: Screening for abdominal aortic aneurysm has been shown to reduce aneurysm-related mortality, but the applicability of the results to the whole of the UK has been questioned. This study examined screening in a remote and rural area. METHODS: Over 3 years, men aged 65-74 years were offered screening in the community by ultrasonography, usually in general practitioner surgeries. Men with an aneurysm were rescanned at intervals or assessed for surgery. The screening and hospital costs of the programme were calculated. RESULTS: Some 9323 men were offered screening of whom 8355 (89.6 per cent) attended. Uptake was high in all areas. A total of 430 scans (5.1 per cent) were abnormal; 40 men had an aneurysm greater than 55 mm in diameter. Twenty further men had an aorta that enlarged to greater than 55 mm during follow-up. A total of 54 men had elective repair with one death (mortality rate 2 per cent). The cost of screening alone was 16 pound per invitation and the overall cost of the programme, including surgery, was 58 pound per invitation. CONCLUSION: Screening for abdominal aortic aneurysm can be carried out in a remote and rural area with high uptake, acceptable clinical results and at no greater cost than in more densely populated areas.

Whole-leg duplex mapping for varicose veins: observations on patterns of reflux in recurrent and primary legs, with clinical correlation.

BACKGROUND: the variability of venous reflux patterns complicate the management of venous disease. Our study investigates specific variations in venous anatomy and patterns of reflux in varying clinical situations. METHODS: prospective analysis of 464 legs in 355 patients was performed by complete duplex venous mapping of both primary and recurrent varicose veins. Hand Held Doppler (HHD) and Duplex Ultrasonography (Duplex US) observations in the popliteal fossa were compared in a subgroup of 89 patients with primary varicose veins. Distribution of venous system disease was correlated with clinical severity in a subgroup of 117 affected legs which was representative of the overall study group. RESULTS: sapheno-femoral junction (SFJ) incompetence predominated in both primary and recurrent varicose veins. Only 21% of primary legs and 25% of recurrent legs had sapheno-popliteal junction (SPJ) incompetence. SPJ incompetence was present in only 42% of cases where reflux in the popliteal region on HHD had been demonstrated. A proportion of both primary and recurrent varicose veins had evidence of deep venous incompetence (DVI). Sixty-four percent of primary leg ulcer patients had superficial incompetence alone. In patients with recurrent varicosities and ulceration, 57% had SPJ incompetence, 64% multiple sites and 50% DVI. CONCLUSION: the complex variations of varicose vein anatomy and functional pathology in the lower limb are currently best assessed by complete whole-leg venous duplex mapping.

World association for the advancement of veterinary parasitology (WAAVP): second edition of guidelines for evaluating the efficacy of equine anthelmintics.

These guidelines have been designed to assist in the planning, operation and interpretation of studies which would serve to assess the efficacy of drugs against internal parasites of horses. Although the term anthelmintic is used in the title and text, these guidelines include studies on drug efficacy against larvae of horse bot flies, Gasterophilus spp., which are non-helminth parasites commonly occurring in the stomach of horses. The advantages, disadvantages and application of critical and controlled tests are presented. Information is also provided on selection of animals, housing, feed, dose titration, confirmatory and clinical trials, record keeping and necropsy procedures. These guidelines should assist both investigators and registration authorities in the evaluation of compounds using comparable and standard procedures with the minimum number of animals.

Ocular manifestations of leukemia: leukemic infiltration versus infectious process.

OBJECTIVE: To determine whether specific guidelines can be developed to distinguish whether retinal infiltration in leukemia patients represents infection or neoplasia. DESIGN: Retrospective noncomparative interventional case series. PARTICIPANTS: Six patients recently seen at University of California San Francisco with retinal infiltrates in a setting of leukemia, for which adequate written and photographic information of disease course was available. INTERVENTION: Observation consisted of retrospective review of clinic charts, hospital medical records, and fundus photographs. MAIN OUTCOME MEASURES: Determination of whether retinal infiltrates represented neoplasia or infection was made by review of medical records. RESULTS: In this series, neoplastic retinal infiltrates were found in patients who had newly diagnosed leukemia and those who were in blast crisis. In contrast, the two patients who were in complete remission, but had undergone bone marrow transplantation, had retinal infiltrates attributable to infection. CONCLUSIONS: Every patient with retinal infiltrates in the setting of newly or previously diagnosed leukemia requires a systemic and central nervous system workup before the initiation of ophthalmologic treatment. The systemic status of the patient is highly informative in determining whether infection or neoplasia is responsible for the infiltration.

Relatedness of Escherichia coli colonizing women longitudinally.

PURPOSE: The longitudinal colonization patterns by Escherichia coli of the vaginal introitus and urinary tract were investigated. MATERIALS AND METHODS: Cultures of the vaginal introitus and midstream urine were collected once a week for 12 consecutive weeks from five women with (patients) and five without (controls) a history of urinary tract infection (UTI). RESULTS: A total of 63 E. coli isolates was obtained from the 10 women, 26 from controls and 37 from patients. The bacterial counts of E. coli present in control individuals were uniformly low, < or = 200 E. coli/mL. The numbers in patients were higher and more variable, reaching > 10(5)/mL in urine and vaginal specimens. In 16 instances, E. coli was present in the urine and the vaginal introitus concurrently (matched isolates). Random amplified polymorphic DNA (RAPD) fingerprinting was used to characterize all matched E. coli isolates. Concurrent vaginal and urinary tract colonization was more common in the patient population, and usually, the same E. coli strain was present at both sites; only 15% of the matched isolates represented different strains. The RAPD fingerprinting was also carried out on selected isolates recovered from four patients and three control individuals over the 12-week study period. Colonization of the vaginal introitus and urinary tract in these individuals varied over time. Generally, however, a predominant E. coli strain was present in the vaginal milieu, urinary tract, or both, either continuously (for as long as 9 consecutive weeks in one patient) or intermittently. CONCLUSION: The results support the concept that the vaginal mucosa acts as reservoir of E. coli which may enter the urinary tract.

Community mortality after ruptured abdominal aortic aneurysm is unrelated to the distance from the surgical centre.

BACKGROUND: Ruptured abdominal aortic aneurysm (RAAA) carries a high community mortality. Raigmore Hospital, Inverness serves Highland Region, an area the size of Wales with a population of 204,000. The aim of this retrospective review was to determine the community mortality and hospital mortality rates from RAAA in Highland Region and to assess whether distance travelled had any significant impact on survival. METHODS: Data were retrieved from hospital records, the Registrar General for Scotland and the Information and Statistics Division of the National Health Service in Scotland about patients diagnosed with RAAA between 1992 and 1999. RESULTS: Of 198 patients with RAAA, 131 (66 per cent) were transferred to Raigmore Hospital while the other 67 (34 per cent) died in a community hospital or at home. Of those reaching Raigmore 109 (83 per cent) had surgery, of whom 65 (60 per cent) survived. The overall community mortality rate was 67 per cent while the hospital mortality rate was 50 per cent. The hospital and community mortality rates for patients living within 50 miles of Raigmore Hospital were 60 and 67 per cent respectively, compared with 26 and 68 per cent for those living more than 50 miles away. CONCLUSION: Distance from Raigmore Hospital had no significant impact on community mortality from RAAA.

Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of Mertk.

The Royal College of Surgeons (RCS) rat is a widely studied animal model of retinal degeneration in which the inability of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segments leads to a progressive loss of rod and cone photoreceptors. We recently used positional cloning to demonstrate that the gene Mertk likely corresponds to the retinal dystrophy (rdy) locus of the RCS rat. In the present study, we sought to determine whether gene transfer of Mertk to a RCS rat retina would result in correction of the RPE phagocytosis defect and preservation of photoreceptors. We used subretinal injection of a recombinant replication-deficient adenovirus encoding rat Mertk to deliver the gene to the eyes of young RCS rats. Electrophysiological assessment of animals 30 days after injection revealed an increased sensitivity of treated eyes to low-intensity light. Histologic and ultrastructural assessment demonstrated substantial sparing of photoreceptors, preservation of outer segment structure, and correction of the RPE phagocytosis defect in areas surrounding the injection site. Our results provide definitive evidence that mutation of Mertk underlies the RCS retinal dystrophy phenotype, and that the phenotype can be corrected by treatment of juvenile animals. To our knowledge, this is the first demonstration of complementation of both a functional cellular defect (phagocytosis) and a photoreceptor degeneration by gene transfer to the RPE. These results, together with the recent discovery of MERTK mutations in individuals with retinitis pigmentosa, emphasize the importance of the RCS rat as a model for gene therapy of diseases that arise from RPE dysfunction.

Saxitoxin: an anesthetic of the deepithelialized rabbit cornea.

PURPOSE: The aim of the current study was to define the efficacy of saxitoxin as a corneal anesthetic in rabbits after mechanical corneal abrasion and photorefractive keratectomy (PRK). METHODS: Twelve Dutch belted rabbits were given a single 1.2-microg dose of saxitoxin or vehicle after mechanical abrasion of the cornea. Corneal sensation was evaluated hourly for 6 hours. A second group of 12 Dutch belted rabbits was given a 1.2-microg dose of saxitoxin or vehicle every 5 hours for 30 hours after PRK. Corneal sensation was evaluated after 5, 10, 15, 20, 25, and 30 hours. Pachometry was performed before PRK and again after the epithelial defects had healed. The rate of epithelial defect closure was assessed by measuring the epithelial defect size 25, 42, 65, 88, and 113 hours after PRK. RESULTS: A dose of 1.2 microg of saxitoxin given every 5 hours produced continuous corneal anesthesia after PRK. There was no difference in the rate of wound healing between eyes treated with saxitoxin and vehicle. There was no difference in the degree of wound healing, as measured by pachometry, between eyes treated with saxitoxin and vehicle. There were no apparent ocular or systemic toxic effects from saxitoxin administration. CONCLUSION: At a dose of 1.2 microg, saxitoxin is a safe, effective, long-acting corneal anesthetic in rabbits after PRK.