Persistent symptoms, mainly joint and muscular pain and depression, have been reported several months after Chikungunya virus (CHIKV) infection. Their frequency and their impact on quality of life have not been compared with those of an unexposed population. In the present study, we aimed to describe the frequency of prolonged clinical manifestations of CHIKV infection and to measure the impact on quality of life and health care consumption in comparison with that of an unexposed population, more than one year after infection.In a retrospective cohort study, 199 subjects who had serologically confirmed CHIKV infection (CHIK+) were compared with 199 sero-negative subjects (CHIK–) matched for age, gender and area of residence in La Réunion Island. Following an average time of 17 months from the acute phase of infection, participants were interviewed by telephone about current symptoms, medical consumption during the last 12 months and quality of life assessed by the 12-items Short-Form Health Survey (SF-12) scale. At the time of study, 112 (56%) CHIK+ persons reported they were fully recovered. CHIK+ complained more frequently than CHIK– of arthralgia , myalgia , fatigue , depression  and hair loss . There was no significant difference between CHIK+ and CHIK– subjects regarding medical consumption in the past year. The mean (SD) score of the SF-12 Physical Component Summary was 46.4 (10.8) in CHIK+ versus 49.1 (9.3) in CHIK– (p = 0.04). There was no significant difference between the two groups for the Mental Component Summary.More than one year following the acute phase of infection, CHIK+ subjects reported more disabilities than those who were CHIK–. These persistent disabilities, however, have no significant influence on medical consumption, and the impact on quality of life is moderate. Aedes mosquitoes . This disease is endemic to many countries in western, central, eastern and southern Africa, Indian Ocean and West Pacific Islands and South-East Asia Chikungunya is a disease caused by an arboviral alphavirus transmitted to humans by Chikungunya virus (CHIKV) is an enveloped, RNA positive-strand Alphavirus belonging to the Togaviridae family Nevertheless, several authors have reported persistent clinical manifestations (mainly joint and muscular pain and depression) several months after acute infection, leading some of them to consider the possibility of chronic forms of Chikungunya infection We conducted an investigation to describe the frequency of late clinical manifestations of Chikungunya virus infection and to measure the impact on quality of life and health care consumption, compared with an uninfected population, in the context of the epidemic occurring in La Réunion Island.Among the 434 people contacted, 27 refused to respond to the questionnaire and 9 were excluded from the analysis because of the impossibility of matching, leaving 199 pairs for analysis. The participation rate was 92% (398 out of 434).The mean age of the study population was 42 years (range: 2–91). Following an average time of 17 months (range: 5–28) from the acute phase of disease, 112 of the 199 CHIK+ subjects (56%) reported that they were fully recovered. The CHIK+ subjects aged under 30 reported a faster recovery than the older subjects  . There wSymptoms at the time of study are shown in There was no significant difference between CHIK+ and CHIK– subjects in the frequency of use of analgesics, medical consultations or hospitalizations in the last 12 months .Quality of life was assessed in all subjects aged 15 or over (N = 324). The average score (SD) of the physical component summary (PCS) was significantly lower in CHIK+ subjects than CHIK– (46.4 (10.8) versus 49.1 (9.3)) .Compared with the CHIK– subjects, the CHIK+ subjects' worst score for the PCS was found in patients aged 30 to 59 and in females . After BHere we report a comparative study of longstanding disabilities owing to CHIKV infection as they are reported within the community. It showed that several subjective symptoms  reported by people infected by CHIKV more than one year after acute infection may be attributable largely (46.4 to 73.7%) to the past infection. The perceived quality of life in these subjects was barely changed, however, as demonstrated by the SF-12. In addition, CHIKV infection was shown to have no significant impact on the use of analgesics, medical consultations or hospitalizations.To the best of our knowledge, no controlled survey had yet attempted to establish the accountability of an arbovirosis, both on the persistence of non-specific symptoms and on their impact on quality of life in a community. Existence of a “chronic form” of the disease following CHIKV infection had been suggested by uncontrolled observational studies previously reported Chronic symptoms following infection with other “arthritogenic” alphaviruses such as Ross River virus and Sindbis-related virus have been reported previously in series of case studies The basis for the musculoskeletal symptoms of Chikungunya disease is still poorly understood A post-infective fatigue syndrome similar to the symptoms reported here by CHIK+ patients  has been shown to persist for six months or more in 12% of persons following a variety of acute infections, including Ross River virus Little attention was paid to scalp lesions in CHIKV infection. However, in the field of alphaviral infections, hair loss is consistent with alopecia encountered in a mice model of Chikungunya infection Quality of life was already assessed with the SF-12 in other rheumatic conditions, such as rheumatoid arthritis, osteoarthritis and fibromyalgia There is no clear explanation why patients under the age of 30 reported faster recovery compared to those older than 30. Older age and severe initial joint pain have been recognized as risk factors for non-recovery Our report has some strengths and limitations. The investigator was not blinded to the serological status of the subjects; participants were questioned about symptoms long after the acute phase of the disease. Also patients were not examined so alternative diagnoses could not be excluded. However, in contrast with previous studies, a standardized health status questionnaire was used and administered by only one investigator, a validated generic quality of health assessment tool was used (the SF-12), a control group was included for comparison and participants were randomly selected through a population-based cohort Whilst there is appreciable temporary disability associated with acute CHIKV infection, and even though more frequent manifestations are reported by infected persons more than a year after the acute phase of the condition, the resulting impact on quality of life and medical consumption seems moderate. Despite alarming signs observed throughout the outbreak in La Réunion, such as reports of severe atypical formsWe conducted a retrospective cohort study to compare subjects who had been infected by CHIKV during the 2005 and 2006 outbreaks in La Réunion Island with those who had not been infected. The study sample was derived from the SEROCHIK survey, a cross-sectional, population-based seroprevalence study aimed at assessing the prevalence of CHIKV infection in the community soon after the 2006 outbreak From this population, we selected pairs of subjects with one subject with serologically confirmed CHIKV infection (CHIK+) and one matched subject with negative serology (CHIK–). Pairs of subjects were obtained by balanced random sampling without replacement from 24 strata defined by a unique combination of age , gender and area of residence .La Réunion is a French overseas department of 787,836 inhabitants located on a volcanic island of 2511 km2 belonging to the Mascarene Islands in the south-western Indian Ocean. Between 2005 and 2006, a Chikungunya outbreak occurred in La Réunion, in which 38.2% of inhabitants became infected Participants were interviewed by telephone between March and June 2007. The questionnaire was administered by one investigator (MKS), using closed questions addressing current symptoms, medical consumption during the last 12 months  and quality of life assessed by the SF-12 scale The patients were questioned about the presence or absence of symptoms on the day of the telephone interview. Illness history was recorded during the interview using questions formulated in lay terms. They were asked if they were currently experiencing the following: joint pain , muscle aches , fatigue, depression, hair loss, skin disorders, sleep disorders or digestive disorders such as nausea, vomiting or diarrhea. When patients reported presence of arthralgia, they were asked to localize the involved joints.The SF-12 explores the physical and mental aspects of quality of life For children under eight, the questionnaire was completed by parents. The SF-12 scale addresses only people of 16 years and over.We considered that an absolute difference of 15% in the frequency of musculoskeletal  symptoms between CHIK+ and CHIK– subjects would be of interest. It has been reported that about 45% of the adult population suffer musculoskeletal pain Assessment of explanatory variables was conducted by univariate analysis. For paired samples, qualitative variables were analysed using exact Mac Nemar's test and continuous variables (SF-12 components) were analysed using Wilcoxon's rank test. Adjusted relative risks (RR) and their 95% confidence interval (95% CI) were calculated for each persisting symptom controlling for stratification criteria  using Mantel-Haenszel methods For the evaluation of quality of life, specific analyses were conducted separately in groups based on sex and age. We then performed five tests adjusted by Bonferroni correction and the statistical significance was set at p = 0.01 with a bilateral formulation.EpiData 3.1™ software  was used for data entry and Stata 10.0™ software  for analysis.During the SEROCHIK survey, which had received ethical approval, the participating subjects had been informed that they might be called back for ancillary research. During the telephone interview, the objectives of the study were presented and oral consent to participate was obtained. For people under the age of 18, the inform consent form was completed by parents.


Background Multiple epiphyseal dysplasia (MED) is a common genetically and clinically heterogeneous skeletal dysplasia characterized by early-onset osteoarthritis, mainly in the hip and knee, and mild-to-moderate short stature. Here we report on a 6-generation MED family with 17 affected members.Method The clinical and radiographic data on the 12 affected members still living were scrutinized. A structured inquiry comprising state of health and MED-related symptoms since birth up to the present time and the osteoarthritis outcome (KOOS) questionnaire were sent to all living family members with MED. The 5 known gene loci for autosomal dominant MED were analyzed for linkage, using fluorescence-labeled microsatellite markers. Linkage was ascertained with markers close to the COL9A2 gene, which was analyzed for mutations by sequencing.Results We identified an exon 3 donor splice mutation in the COL9A2 gene in all affected family members. Clinical, radiographic, and questionnaire data from affected family members suggested that MED caused by COL9A2 mutations starts in early childhood with knee pain accompanied by delayed ossification of femoral epiphyses. The disease then either stabilizes during puberty or progresses with additional joints becoming affected; joint surgery might be necessary. The progression of the disease also affects muscles, with increasing atrophy, resulting in muscle fatigue and pain. Muscular atrophy has not been reported earlier in cases with COL9A2 mutations.Interpretation In a patient with clinically suspected or verified MED, it is important to perform DNA-based analysis to identify a possible disease-causing mutation. This information can be used to carry out genetic risk assessment of other family members and to achieve an early and correct diagnosis in the children. Multiple epiphyseal dysplasia (MED) is an autosomal dominant skeletal dysplasia that affects approximately 1 in 10,000 individuals. It was first described in 1937, by the Swedish radiologist Ribbing. The predominant features of the disease are delayed and irregular ossification of epiphyses and early onset of osteoarthritis. The MED symptoms appear during early childhood, usually with pain in the knees after exercise. Children who are affected often have difficulty in getting up from the floor and have a waddling gait. They frequently complain of fatigue during long walking. Adult height is usually in the lower range of normal. The limbs are relatively short in comparison to the trunk. Pain and joint deformities progress with age, resulting in early-onset osteoarthritis, particularly of the large weight-bearing joints. The spine is usually normal . The diaCOMP, which encodes cartilage oligomeric matrix protein; COL9A1, COL9A2, and COL9A3, which code for the 3 alpha chains of cartilage-specific type IX collagen; and MATN3, which encodes matrilin-3, a cartilage extracellular matrix protein. COMP is mutated in 80% of all MED samples analyzed. In approximately 10–20% of all samples tested, a mutation cannot be identified in any of the 5 known genes mentioned above, suggesting that mutations in other hitherto unidentified genes are also involved in the pathogenesis of dominant MED ((DTDST). This form of MED is characterized by malformations of the hands, feet, and knees, with a double-layered patella and scoliosis  for autosomal dominant MED were genotyped using fluorescence-labeled microsatellite markers and 25 ng of DNA per PCR reaction, as described previously  and an ABI PRISM 3730 DNA analyser (Applied Biosystems). The DNA sequences obtained were analyzed with Sequencher v.4.1.2 .Blood samples were collected from 24 family members  after obtaining informed consent. Genomic DNA was extracted from peripheral lymphocytes according to standard procedures. The 5 known gene loci eviously . Genotypeviously . The COLThe presenting symptoms of the 12 living family members with MED were pain in the knees and fatigue during long walks at 2–7 years of age . The maiThe disorder varied strikingly within the family. For example, a 95-year-old male patient (III:2) had a relatively mild form of the disease with only slight pain in the knees during early childhood. He was then free of symptoms until the age of 27, after which he had a few episodes of pain in the knees caused by osteochondritis dissecans. No surgery was needed. By the age of 60 he suffered from slight pain in the hips, and this pain began to increase at the age of 88, requiring bilateral hip replacement when he was 93. In contrast, a 93-year-old female patient (III:6) had a severe form of MED. From the age of 4, she had pain in the knees when weight bearing and difficulty in walking. At the age of 14 she began to suffer from dull ache in the knees. Knee surgery with removal of loose cartilaginous fragments was performed when she was 19 years old, and again at the age of 85. After the age of 35 she had increasing, disabling pain in several joints and in muscles. She had total hip replacements at the age of 65 and at 76. At the age of 85 severe muscular atrophy developed, with motor disability, and she became confined to a wheelchair.The 8 females affected had a more severe form of MED than the 4 males, and together they had undergone 11 orthopedic operations. Only 1 of the 4 males needed orthopedic surgery. All the adult family members affected had a university education and full-time employment.COMP, MATN3, COL9A1, and COL9A3 loci. A positive linkage to the disease was obtained with marker D1S1598 close to the COL9A2 gene (maximum LOD score of 2.7). The COL9A2 gene was analyzed for mutations by sequencing of exon 3 and intron 3—the part of the gene where mutations have been identified in previous MED patients  chance that each child of an affected parent will inherit the mutation and thus be affected with the disorder. The exons of the COL9A2 gene are translated into protein (a subunit of collagen IX), while the introns of the gene are removed and not translated. When the mutation is found at the donor splice site, it means that it is located right at the site of the cutoff between the exon and intron. COL9A2gene; this type of MED is defined as EDM2 (COL9A1 and COL9A3, COL9A2 forms the collagen IX heterotrimer. Previous studies suggest that collagen IX functions as a bridge between collagen fibrils and matrix macromolecules in the joint cartilage (All of the affected family members in our study were found to carry an identical mutation at the donor splice site of exon 3/intron 3 of the  as EDM2 . The EDM as EDM2 . The COL as EDM2 . Togetheartilage .COMP and COL9A3 genes (In addition to the genetic heterogeneity in MED, there is a marked intrafamilial variability in the clinical phenotype, suggesting that environmental and/or modifying genetic factors also play a role . DespiteA3 genes .In a patient with MED, it is important to carry out a DNA-based analysis to identify a possible disease-causing mutation. This information can be used in genetic risk assessment of other family members and to achieve an early and correct diagnosis in children in the family with suspected MED. The risk for each child of being an affected person is 1 in 2, whereas the risk for the offspring of an unaffected person is insignificant. There is as yet no curative treatment for the disease, but much can be done to alleviate the symptoms, counteract deformities, and compensate for functional limitations. Families that have children with MED need to be in early contact with a pediatric orthopedic surgeon; operations may be necessary to minimize joint deformation and to preserve mobility. The families should also be connected to a center for pediatric and youth rehabilitation at an early stage. The occupational therapist can work out techniques to help the child to manage everyday activities at school and at home. The physiotherapist is responsible for evaluation, treatment, and programs for motor training. The various therapeutic components should be co-ordinated to achieve the best possible results. Adults with MED need to continue to interact with an orthopedic surgeon, an occupational therapist, and a physiotherapist, and sometimes with a rehabilitation center for adults. Joint replacement may become unavoidable with age.


Acute cholecystitis occurs frequently in the elderly and in patients with gall stones. Most cases of severe or recurrent cholecystitis eventually require surgery, usually laparoscopic cholecystectomy in the Western World. It is unclear whether an initial, conservative approach with antibiotic and symptomatic therapy followed by delayed elective surgery would result in better morbidity and outcome than immediate surgery. At present, treatment is generally determined by whether the patient first sees a surgeon or a gastroenterologist. We wish to investigate whether both approaches are equivalent. The primary endpoint is the morbidity until day 75 after inclusion into the study.A multicenter, prospective, randomized non-blinded study to compare treatment outcome, complications and 75-day morbidity in patients with acute cholecystitis randomized to laparoscopic cholecystectomy within 24 hours of symptom onset or antibiotic treatment with moxifloxacin and subsequent elective cholecystectomy. For consistency in both arms moxifloxacin, a fluorquinolone with broad spectrum of activity and high bile concentration is used as antibiotic. Duration: October 2006 – November 2008The trial was planned and is being conducted and analysed by the Departments of Gastroenterology and General Surgery at the University Hospital of Heidelberg according to the ethical, regulatory and scientific principles governing clinical research as set out in the Declaration of Helsinki (1989) and the Good Clinical Practice guideline (GCP).ClinicalTrials.gov NCT00447304 Acute cholecystitis is one of the most significant acute diseases in the Western World, and may be associated with only mild pain and nausea or become a severe, life-threatening illness due to complications. Acute cholecystitis is mainly caused by gall stones, whilst cholestasis is mainly associated with superinfection with bacteria, in general species of enterobacteria, enterococci, bacteroides and anaerobic streptococci .The principal complication is recurrent biliary colic and cholestasis. The latter may lead to ascending cholangitis, and whilst this can be managed with antibiotics, other complications can not be cured conservatively, such as gangrenous changes, gall bladder perforation and biliary leakage, and acute necrotic gallstone pancreatitis -5. LiverThe risk of developing second and subsequent episodes of acute cholecystitis is higher than the risk of suffering an initial episode ,8. LaparThe approach taken is often decided by whether the patient first sees a gastroenterologist, who favors conservative initial antibiotic therapy with later elective surgery, or a surgeon, who favors immediate surgery.It is still unclear which approach is better in medical and health economic terms. The infection may not respond to conservative treatment on one hand, on the other hand surgical intervention while the disease is acute may increase complications, and conversion to open surgery may be necessary. It is unclear whether it is better to conduct early cholecystectomy, thereby avoiding the risk of recurrent cholecystitis or pancreatitis.A meta-analysis by Papi et al. (2003) including 12 prospective randomized trials showed no significant difference for morbidity and mortality between immediate surgical intervention (laparoscopic or open) and elective surgery after the acute inflammation had subsided . The numAll studies in the meta-analysis had been performed between 1970 and 2000, and Papi concluded that new studies in an adequate number of patients to show statistical significance should be performed . Two subIn many centers early cholecystectomy is well established although the evidence is not yet conclusive. To our knowledge there has never been a study in which both specialties – gastroenterology and surgery – are equally involved.®) covers the spectrum of gram-positive, gram-negative and anaerobic bacteria usually responsible for intra-abdominal infections [Moxifloxacin  laparoscopic cholecystectomy within 24 hours of hospital admission; and (ii) initial antibiotic treatment with moxifloxacin followed by cholecystectomy in the infection-free interval (Day 7 to 45).The trial is a GCP-compliant, multicenter, prospective, randomized non-blinded study. Patients with acute cholecystitis meeting the inclusion criteria are randomized to one of the treatment arms. The study is being audited by members of a contract research organization (CRO) and may be subject to government inspection. The trial was approved by the German authorities and Ethical committees.M1 - ρM2 /> 0.1, where ρMi is the morbidity rate of treatment group i. Complications are expected in 16% of patients in each group; each group therefore needs to enroll 273 patients to permit verification of the null-hypothesis with an α-error of 0.05 and a β-error at 0.15, yielding a power of 90%. Assuming a validity rate of 85%, 322 patients are required per group, resulting in a total patient sample of 644.The primary aim of the study is to compare morbidity in the two groups 75 days after enrolment. A difference in morbidity of less than 10% is defined as equivalent. The null-hypothesis is /ρ• Age ≥ 18 years• Patients with acute cholecystitis with three of the following symptoms or signs• Abdominal pain in the upper right quadrant• Murphy's sign3/μl• Leukocytosis > 10 × 10• Rectal temperature > 38°C or < 36.5°Cplus• Cholecystolithiasis (stones/sludge) or sonographic signs of cholecystitis • Immediate antibiotic therapy (400 mg Moxifloxacin i.v. once a day)• Laparoscopic cholecystectomy possible within 24 hours after presentation of the patient• Informed consent• ASA IV and V table • Septic shock• Perforation or abscess of the gall bladder• No possibility of laparoscopic surgery• Additional antibiotics needed for secondary disease• Intolerance to moxifloxacin or other quinolones• Pregnancy , breast feeding• Life-expectancy < 48 hours• End-stage liver disease (Child-Pugh C)• Psychiatric or severe neurologic disease• Relevant bradycardia or other symptomatic arrhythmias• Significant cardiac disease• Disorder with QT prolongation• Hypocalcaemia or other electrolyte disorders• Earlier participation in this trialThe final protocol was approved by the independent ethics committee of the University of Heidelberg. The study was registered at ClinicalTrials.gov (NCT00447304). Patients who are scheduled for laparoscopic cholecystectomy (immediate or elective) due to acute cholecystitis are informed about the trial  and are given the opportunity to participate at the screening visit. Informed consent is required. Patients may withdraw from the study at any time without giving reasons and without jeopardizing their further treatment. The investigator may also withdraw patients if this is in their best interests.This study is randomized to minimize bias. Sealed randomization envelopes are provided in packs of four by the CRO and are held centrally at each investigational site. An envelope is opened when a patient agrees to take part and patients are informed whether they are to be treated with immediate surgery or initial conservative antibiotic therapy.Day 1 is defined as the day the patient presents to the hospital. He undergoes a physical examination, vital signs are documented, and an abdominal ultrasound investigation is performed to confirm the diagnosis of acute cholecystitis. A blood sample is also taken for standard laboratory diagnosis . All relevant concomitant diseases  indicative for morbidity and mortality are recorded .The patient is informed about the trial and is given the opportunity to participate. After the patient has given his informed consent, he is randomized, and all baseline findings, date of birth, age, sex, medical history, height and weight are documented in the CRF.All patients are examined daily while in the hospital. The laboratory investigations are repeated on Day 3. Samples are taken for microbiological cultures, if necessary. At Day 75 (test-of-cure visit), all diagnostic procedures and treatments between Day 1 and Day 75 are documented. The laboratory determinations and physical examination are repeated, and vital signs are measured. Morbidity is documented according to Table • Laparoscopic cholecystectomy in the 24 hours after hospital admission• Antibiotic therapy with moxifloxacin 400 mg i.v. once per day for 48 hours followed by oral Moxifloxacin 400 mg daily or discontinuation of antibiotic treatment if possible• Discharge of the patient as soon as possible after Day 2, if the body temperature, CRP and leukocytes are normal• Test-of-Cure visit at Day 75• Therapy with i.v. moxifloxacin 400 mg once daily for 48 hours followed by oral moxifloxacin 400 mg per day. Discontinuation of moxifloxacin after Day 7, provided body temperature, CRP and leukocytes are normal• Discharge of the patient as soon as possible after Day 4 on oral moxifloxacin• Elective cholecystectomy between Days 7 and 45 after admission to study using single-shot moxifloxacin i.v. for prophylaxis• Test-of-Cure visit at Day 75Primary endpoint is morbidity at the test-of-cure (TOC) visit  in the tested population valid for efficacy.1. Morbidity over 75 days using the scoring system showed in Table 22. Morbidity 3 days after cholecystectomy (immediate and elective)3. Rate of conversion from laparoscopic to open surgery4. Change of antibiotic due to non-response or non-toleration of moxifloxacin5. Mortality at Day 756. Cost-efficiency7. Hospital time8. Safety and tolerance of moxifloxacin9. Duration of hospital stay after cholecystectomy (days)Adverse events and serious adverse events and deaths occurring up to Day 75 were recorded. An adverse event (AE) is every medical event that worsens or impairs the well-being of the patient not being part of the natural course of the disease but may be due to treatment or drug application. The term AE can cover any sign, symptom or reaction, including not normal laboratory findings, independent if caused by the tested procedure and medication or not. Adverse event intensity  and relationship to the treatment or the study drug moxifloxacin  were categorized. Serious adverse events (SAEs) included those events that were fatal, life-threatening, required hospitalization, resulted in disability or otherwise endangered the patient.All AEs and SAEs will be documented in detail in the case report form (CRF) and will be reported to the principal investigator at regular intervals. SAEs have to be reported within 24 hours to the principal investigator, and must be documented separately on an SAE report form within 24 hours. According to law and guidelines, SAEs have to be reported to the ethics committee(s) and supervisory board(s) as necessary. The period of observation for AE reporting is from Day 1 to Day 75.All statistical tests will be performed two-sided with a level of significance of 5%. The patients will be analyzed as treated. The principle analysis will be on evaluable patients and a supportive intent-to-treat analysis will be performed. Patients will be stratified according to severity of their condition (ASA ≤ 2 vs. ASA > 2); the principle analysis will be stratified, but a non-stratified analysis will also be performedThe groups will be tested for equivalence of distribution of age, sex and body mass index. The analysis will be conducted using analysis of variance with the factors study group and severity of disease, stratified by the Cochran-Mantel-Haenszel test.95% confidence intervals (CI) for the difference of morbidity rates will be calculated. If and only if this CI lies between -10% and +10% the two procedures will be considered equivalent. The calculation of the 95% CI will be stratified by severity (ASA ≤ 2 vs. ASA > 2). A Breslow-Day test will be performed to check for homogeneity between these two strata.Amongst the secondary variables, the mean morbidity score in the two groups stratified by severity of disease will be compared using the Wilcoxon rank-sum test. Where appropriate, descriptive statistics will also be performed for all other variables.All eligible patients are seen by a gastroenterologist or surgeon and are enrolled after giving informed consent. The incidence of patients with acute cholecystitis ranges from 10 to >100 per year at different investigational sites. With about 30 sites, it is estimated that enrollment of 644 patients will take about 24 months. All findings are recorded in the patients medical records and CRF provided for this study by the investigator. Data verification is performed by the CRO, who will also perform the analysis on the locked database after plausibility testing and data query correction.The author(s) declare that they have no competing interests.KW participated in the design and coordination of the study and drafted the manuscript. JE, CG and JK participated in the design and coordination and helped to draft the manuscript. JE and CG conceived the study. JE, CG, JK and KW supervised the coordination of the different trial centers. MWB and WS supervised the coordination of the study. All authors read and approved the final version of the manuscript.


In contrast to the large number of studies in children, there is little information on the contribution of genetic factors to Attention Deficit Hyperactivity Disorder (ADHD) in adults.To estimate the heritability of ADHD in adults as assessed by the ADHD index scored from the CAARS .Phenotype data from over 12,000 adults  registered with the Netherlands Twin Register were analyzed using genetic structural equation modeling.Heritability estimates for ADHD from the twin-family study.Heritability of ADHD in adults is estimated around 30% in men and women. There is some evidence for assortative mating. All familial transmission is explained by genetic inheritance, there is no support for the hypothesis that cultural transmission from parents to offspring is important.Heritability for ADHD features in adults is present, but is substantially lower than it is in children. It is associated with considerable social, family, behavioral and cognitive dysfunction and is comorbid with depression, bipolar disorder, anxiety, and substance use A recent study observed strong familial clustering of ADHD in first-degree relatives of adults with ADHD. Compared to a control group adults with ADHD had significantly more first-degree relatives with ADHD (28% versus 5%) In this manuscript we present a quantitative genetic study of adult ADHD in a large sample of twin families who are registered with the Netherlands Twin Register Heritability was estimated using data from twins, their parents and siblings with structural equation modeling and under different models of familial resemblance. Adding data from siblings to the classical twin design results in an increase in power to detect non-additive genetic effects The classical twin design can inform on the influence of shared environment, but is not informative with respect to how much of the environment that is shared among offspring is transmitted from parents to offspring . It is easily conceivable that growing up with a parent who scores high on a number of ADHD features influences the phenotype of their offspring. By adding phenotypic data from parents to the classical twin design, vertical cultural transmission, reflecting the non-genetic influence of the parents' ADHD features on their offspring, can be examined. Because ADHD is expected to have a heritable component, vertical cultural transmission will induce genotype-environment correlation Phenotype data were collected in participants from the Netherlands Twin Register (NTR) by survey in 2004–2005 (survey 7) and in an ongoing study (survey 8) that started in 2009. Data collection for survey 7 has been detailed in Distel et al The study was approved by the Central Ethics Committee on Research involving human subjects of the Vrije Universiteit Amsterdam, an Institutional Review Board certified by the US Office of Human Research Protections . All subjects provided written informed consent.In survey 7, the screening self-report (CAARS - S:SV) form of Conners' Adult ADHD Rating Scales (CAARS) was included that consists of 30 items accessing ADHD symptom level according to DSM-IV criteria Twin-family studies make use of the different degree of genetic relatedness of family members to estimate the relative contribution of genes and environment to the variance of a trait. Monozygotic (MZ) twins are genetically (nearly) identical. Under random mating dizygotic (DZ) twins and non-twin sibling share on average 50% of their segregating genes, while parents and offspring share exactly 50% of their autosomal genes. In quantitative genetic analyses, phenotypic variance is modeled as a function of genetic (G), shared (C), and non-shared environmental (E) influences. Genetic variance can be additive (A), indicating that the effects of alleles are additive, or non-additive  referring to the interaction among alleles. All genetic effects are shared by MZ twins. DZ twins and siblings are correlated 0.5 for their additive genetic values, as are parents and offspring. DZ twins and siblings are correlated 0.25 for their non-additive genetic values, for parents and offspring this correlation is zero. Under non-random mating the correlations among family members for additive genetic values may increase Genetic analyses were carried out using structural equation modeling in Mx 2 with degrees of freedom (df) equal to the difference in the number of parameters in the two models. If the χ2-test yields a significant p-value (e.g. lower than 0.05 or 0.01), the constrained model is deemed not significantly worse. However, the chi-squared value is inflated as the sample size increases. For this reason, big discrepancies in small samples may not be significant whereas small differences in big sized samples are significant. Given the large sample size in this study  decisions about goodness-of-fit were based on the RMSEA (Root Mean Square Error of Approximation) which is obtained as: RMSEA = √(F0/df), where F0 is the chosen fit function The standard approach to the comparison of different models is by means of likelihood-ratio tests, by subtracting the negative log likelihood (-2LL) for a more restricted model from the -2LL for a more general model. This yields a statistic that is distributed as χTest-retest data were available for 2,519 subjects who took part in NTR surveys 7 and 8. The average age was 41.18 (SD = 13.66) at the first and 45.01 (SD = 13.77) at the second assessments. The correlation between the two assessments was 0.66. There were no differences in average scores for ADHD index (7.75 (SD = 3.79) and 7.54 (SD = 3.87)) between the two assessments. The six month test -retest correlation assessed on 200 unrelated individuals who took part in survey 7 was 0.67.The estimates of means from a saturated model (corrected and uncorrected for age) are given in The pattern of familial correlations in Model comparisons  and the offspring generations (average age 34 years). In fact, the parent-offspring correlations were of similar magnitude as the sib-sib and DZ twin correlations, providing evidence that the same set of genes may be playing a role across the adult life-span.The heritability estimate in adults is substantially lower than the heritability of ADHD and Attention Problems in children. A large longitudinal study of children aged 3–12 year estimated heritability for Attention Problems at 75% at all ages The age at which heritability decreases from the high estimates in childhood to the lower estimate as reported in the current study may be somewhere during adolescence. A longitudinal study in young adult twins (18–30 years) of self-reported Attention Problems estimated its heritability at 40% The large difference between childhood and adult ADHD heritability is remarkable if we compare these results to those for IQ and cognition. ADHD tends to be associated with a lower IQ This study analyzed data from over 12,000 adult subjects. Although the large sample size offered the possibility to test for different models of genetic and cultural inheritance, a limitation of the present study is that the large sample size precluded a clinical diagnosis of the phenotype. Another limitation might be that survey studies in volunteer samples increasingly suffer from non-response. Distel et al. It has been speculated that the heritability of ADHD, especially of the persistent form, may be higher in adulthood than in childhood This study clearly established the moderate stability and heritability of ADHD in adults. It paves the way for future investigations needed to fully understand the complex architecture of ADHD, such as studies on the heterogeneity of the phenotype, genome-wide linkage and association studies in adults and investigations of the effects of genotype-environment interaction and correlation. As detailed by recent reviews and meta-analyses


PTPN11, which alters frame, results in premature translation termination, and co-segregates with the phenotype. In a second metachondromatosis family, we confirmed our result by identifying a nonsense mutation in exon 4 of PTPN11 that also co-segregates with the phenotype. Sequencing PTPN11 exon 4 in 469 controls showed no such protein truncating variants, supporting the pathogenicity of these two mutations. This combination of a new technology and a classical genetic approach provides a powerful strategy to discover the genes responsible for unexplained Mendelian disorders.Although more than 2,400 genes have been shown to contain variants that cause Mendelian disease, there are still several thousand such diseases yet to be molecularly defined. The ability of new whole-genome sequencing technologies to rapidly indentify most of the genetic variants in any given genome opens an exciting opportunity to identify these disease genes. Here we sequenced the whole genome of a single patient with the dominant Mendelian disease, metachondromatosis (OMIM 156250), and used partial linkage data from her small family to focus our search for the responsible variant. In the proband, we identified an 11 bp deletion in exon four of  PTPN11, which results in premature translation termination and co-segregates with the phenotype. In a second metachondromatosis family, we identified a nonsense mutation in exon 4 of PTPN11 that also co-segregates with the phenotype. Germline gain-of-function missense mutations in PTPN11 cause an overlapping but distinct group of dominant disorders with involvement of the face, heart, skeleton, skin, and brain, including Noonan syndrome (OMIM 163950), Noonan-like disorder with multiple giant cell lesion syndrome (OMIM 163955), and LEOPARD syndrome (OMIM 151100). Nonsense mutations in PTPN11 have not been described in humans and the loss-of-function PTPN11 mutations we report here are the first to be described in human disease.Metachondromatosis (MC) is an autosomal dominant condition characterized by exostoses (osteochondromas), commonly of the hands and feet, and enchondromas of long bone metaphyses and iliac crests. MC exostoses may regress or even resolve over time, and short stature is not characteristic of MC. Here, we sequenced the whole genome of a single patient with MC and used partial linkage data from her small family to focus our search for the responsible variant. In the proband, we identified an 11 bp deletion in exon four of  Elucidation of the molecular bases of Mendelian disease has provided a rich resource for understanding genetic mechanisms, protein functions, the behavior of biological systems and mechanisms of disease http://www.hgmd.cf.ac.uk/ac/index.php> are in regulatory sequences and this is likely to be an underestimate given the traditional strategies used for mutation detection (largely PCR amplification and sequencing of exons). The same database lists about 7.5% of disease variants as structural variants. WES also requires higher average coverage levels than WGS, both because of the variable success of capturing different regions and because of “allelic imbalance” where one allele is preferentially captured over the other. For these reasons we elected to employ a WGS strategy.The recent development of massively parallel DNA sequencing technologies has reduced the cost and increased the throughput of large-scale sequencing (LSS) and provides a new and potentially powerful way to identify virtually all of the mutations responsible for Mendelian disorders 6). In addition to these approaches, it also is possible to utilize classical genetic strategies that depend on family structure and inheritance patterns to prioritize certain genomic regions. In our case, we show that it is possible to combine partial linkage information with other criteria for prioritizing variants to identify the genetic basis of a rare autosomal dominant disorder .Both WES and WGS identify a large number of sequence variants when compared to the reference sequence, making it important to prioritize variants. This can be based upon both the assessment of the likelihood of the variants being functional, especially in the WGS setting , and on their frequency in healthy control populations. These approaches were used in the WES study that discovered the gene for Miller syndrome (OMIM 263750) by identifying functional mutations in the same gene in each of four unrelated patients and no controls , is an autosomal dominant condition characterized by exostoses, commonly of the bones of the hands and feet, and enchondromas of the metaphyses of long bones and iliac crest. It was first described by Maroteaux in 1971, based on clinical observation of the six affected individuals from two families At the outset of our studies, we had access to a single MC family (Pedigree 1) and had not personally examined individual III-1, who was reported to be unaffected. We performed linkage analysis using multiple individuals from Pedigree 1  and founPTPN11  from Pedigree 1, to an average of 31.8× coverage. In the 42 Mb of candidate regions defined by our linkage results, 95% of the exonic sequence was covered at a depth of >10×. We also sequenced eight unrelated controls and used data available from dbSNP. In the 7p14.1 (14 RefSeq genes), 8q24.1 region (27 RefSeq genes), 2p25 region (20 RefSeq genes), 5q12.1 region (7 RefSeq genes), and 9q31.1–q33.1 (71 RefSeq genes) regions we found no variants unique to the patient genome with a high likelihood of functional significance (stops gained and frameshifting indels). However, in the 12q23 region (105 RefSeq genes), we identified one frameshifting indel, an 11 bp deletion extending to but not including the 3′ base in exon 4 of PTPN11 deletion with the affected status in members of Pedigree 1 using a PCR assay  was also affected with an exostoses of her right proximal tibia.We confirmed the segregation of the PTPN11 deletion was responsible for MC in Pedigree 1. To test this hypothesis, we identified a second family segregating MC as an autosomal dominant trait (Pedigree 2)  . We sequPTPN11 exon 4 in 469 control, unrelated individuals of whom 60% were of European descent similar to that of our two affected families, 11% were African-American, 11% were East Asian and 18% were of other ethnicities. We found no examples of either of these mutations, nor any other variants in exon 4 predicted to result in the loss of PTPN11 function.As a further test of the significance of the mutations we identified, we amplified and successfully Sanger sequenced PTPN11 encodes the protein tyrosine phosphatase SHP-2, which is an src homology-2 (SH2)-containing protein tyrosine phosphate (PTP) that is highly conserved among metazoans and plays a central role in RAS/MAPK signaling downstream of several receptor tyrosine kinases including EGFR and FGFR PTPN11 cause an overlapping but distinct group of dominant disorders with involvement of the face, heart, skeleton, skin and brain, including Noonan syndrome (OMIM 163950), Noonan-like disorder with multiple giant cell lesion syndrome (OMIM 163955) and LEOPARD syndrome (OMIM 151100) PTPN11 have not been described in humans, but in mice a gene-targeted mutant Ptpn11 allele that deletes codons 46–110 is an early developmental recessive lethal Ptpn11+/− mice PTPN11 mutations we report here (c.514_524del11 and p.R138X) are the first to be described in human disease. Not surprisingly, MC has essentially no phenotypic overlap with the other disorders caused by PTPN11 mutations, with the possible exception that one affected individual in Pedigree 2, I-2, a 71-year-old male, has multiple truncal lentigenes  and the trichorhinophalangeal syndrome type II (OMIM150230). Additionally, it is interesting to speculate on the focal nature and limited duration of the enchondromas and exostoses in MC. The local nature and childhood onset of these lesions suggests the possibility that a second mutational event is required for their appearance, a possibility that can be tested by examining PTPN11 in these tumors. The reason for spontaneous regression of some of these lesions is unclear, but may be due to the developmental maturation of the affected tissue.Our results suggest that Our study adds to a small but growing list of examples where genome-wide sequencing approaches have successfully identified rare, high-penetrant risk factors for disease. Ours is one of the first to take the whole-genome approach, although the variant we identified would have been found using either WGS or WES. However, two key distinguishing features of this study are that discovery of the disease-causing variant resulted from the initial sequencing of only a single patient genome and that weak linkage evidence helped to identify regions most likely to harbor the causative variant. The linkage evidence present in this family restricted our search for the causal variant to 42 Mb, which contained only one protein-truncating variant unique to our sequenced case. Without this linkage evidence, sifting through the 109 protein-truncating variants unique to this genome  to find the causal variant would have been a difficult task. It would also have been difficult to sift through the many unique variants in the linked regions if it were not possible to assess the function of all identified variants, whether previously known or novel as the one we found. We also note that generating the necessary sequence data for all genes in the 42 Mb implicated region in the first family would be a daunting task using traditional sequencing approaches. This paradigm of WGS with the prioritization of variants by predicted functional consequence and frequency in controls combined with information gleaned by classical genetic approaches should prove effective for other unexplained Mendelian phenotypes and may also prove effective in the study of more common diseases that show modest linkage evidence.Informed consent was obtained through a Johns Hopkins Medical Institutions IRB approved protocol and a Duke University IRB approved protocol.Individuals with MC from two families were identified and recruited from the Johns Hopkins Hospital Genetics Clinic. Informed consent was obtained through a Johns Hopkins Medical Institutions IRB approved protocol. Family 1 was a four-generation family comprised of 12 individuals for whom we had DNA. Seven individuals were classified as “affected” and five were “unaffected”. Family 2 was a three-generation family of three “affected” individuals and seven “unaffecteds” for whom we had DNA.In preparation for interpreting the results of our WGS, we performed linkage analysis on Pedigree 1  by genothttp://www.svaproject.org/) to group all variants into functional categories. We expected that the variant responsible for MC would be rare and would not be found in dbSNP or in the control sequenced genomes. We also expected that it would be a severe functional variant, and thus concentrated on those variants that resulted in protein truncation: nonsense mutations and frameshifting indels.We performed WGS on a single individual (V-1) from MC pedigree 1 with an Illumina Genome Analyzer II. The sequence runs were paired-end 75 base pair reads, with 110 billion bases passing the Illumina analysis filter. To assess overall coverage, all gaps (stretches of N's) in the reference genome (Ensembl build 36 release 50) were excluded, resulting in the reference having 2,855,996,286 bases. After accounting for PCR duplicates and reads that did not align to the reference genome, our genomic coverage was 31.8×. We defined a “covered” base as one with at least five reads with a Phred-like consensus score greater than zero. Using these criteria, we covered approximately 97.8% of the reference genome. We then used data from eight control genomes that we had sequenced to an average coverage of 35.9× and dbSNP as filters to remove common variants. To identify candidate pathogenic mutations, we used SVA (Text S1Supplementary methods and figures.(3.28 MB DOC)Click here for additional data file.


Dictyostelium mucoroides-7 (Dm7) exhibits clear dimorphism; macrocyst formation as a sexual process and sorocap formation as an asexual process. These two life cycles are regulated by two regulators, ethylene and cyclic AMP (cAMP). This is the first report demonstrating a novel function of ethylene at the cellular level. That is, ethylene induces a zygote formed by cell fusion and subsequent nuclear fusion. Recently, the function of ethylene at the molecular level has been clarified as it induces zygote formation through an enhanced expression of a novel gene, zyg1. The signaling pathway for induction or inhibition of zygote formation is now trying to be clarified focusing on the ZYG1 protein.The cellular slime mold,  The cellular slime mold is a microorganism living in the soil. It has some interesting features. The amoeboid cells grow and multiply feeding on bacteria as a food supply at the vegetative growth phase. Upon exhaustion of the bacterial food supply (starvation), however, starving cells gather together by chemotactic movement toward cAMP, forming aggregation streams. A tip is formed on the top of each cell aggregate, which then migrates as a slug-shaped mass. After the migration, the slug changes dramatically its shape to form a sorocarp consisting of a stalk with an apical mass of spores. Thus, the differentiation phase of the cellular slime mold is separated from the growth phase. Furthermore, they have only two cell types of differentiation, that is, stalk and spore cells. These characters are quite suitable for studies of differentiation including the mechanisms of the transition from growth to the differentiation phase. As they are usually haploid, it is easier to manipulate genes, such as clone, knockout genes and so on. For this reason, the cellular slime mold is regarded as a model organism.Dictyostelium mucoroides 7 (Dm7), one of the species of cellular slime molds, forms macrocysts homothallically. Macrocyst formation in Dm7 is characterized by the formation of large aggregates after starvation, which are subdivided into smaller masses (precysts), each of which is surrounded by a fibrillar sheath. At the center of each precyst there arises a cytophagic cell (giant cell), which in turn engulfs all the other cells in the precyst. The engulfed cells (endocytes) are eventually broken down into granular remnants. The enlarged cytophagic cell finally becomes surrounded by a thick wall to form the mature macrocyst , the wild-type NC4 cells undergo to mate with an opposite mating type, V12M2 cells heterothallically. Dm7, one of the strains of Dictyostelium mucoroides (Dm) has not a mating type and mates by itself homothallically. Since RaperPolysphondylium violaceum (Pv)D. giganteum (Dg),P. pallidum (Pp) and Dd.Macrocyst formation was first reported by Brefeld in 1869.Recently, giant cells formed during macrocyst formation in Dm 7 have been proved to be zygotes that are produced by cell fusion and subsequent nuclear fusion.2) and charcoal-absorbed gases (CAG), as candidates of chemical regulators in Dm7. When the gases were absorbed by charcoal, only sorocarps were formed instead of macrocysts.2 gas with KOH, CO2 could be an antagonist of CAG. Underwater favors macrocyst formation in contrast to sorocarp formation which predominantly takes place in mineral oil. This suggested that CAG might have some hydrophobic characters. Based on the idea that a hydrophobic gas (es) produced by cells may accumulate around cells at higher levels due to its slow diffusion into water and act antagonistically to CO2, we speculated on ethylene as a potent candidate of CAG. The possibility that ethylene might actually act as CAG was tested using MF1 cells, a spontaneous mutant isolated from Dm7. The mutant MF1 cells are able to form macrocysts even in the light depending upon cell densities plated. This suggested that CAG might have the threshold concentration to induce macrocyst formation. In fact, MF1 cells failed to form macrocysts even at higher cell densities, when they were allowed to develop in a larger incubation chamber. It was supposed that the concentration of CAG produced by the cells would be insufficient for induction of macrocysts by being diluted in the larger chamber. However, MF1 cells changed their developmental forms from sorocarps to macrocysts even in the larger chamber by the addition of ethylene. As was expected, inhibitors of ethylene biosynthesis, such as aminooxyacetic acid (AOA) and aminoethoxyvinyl glycine (AVG) greatly inhibited macrocyst formation. Ethylene production by MF1 cells and Dm7 cells was confirmed by gas chromatography.Dd-aco, an 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase homologue gene, isolated from Dd  was introduced into Dm7 cells to produce transformants. Transformant over-producing Dd-aco (ACOOE) actually produced a larger amount of ethylene than the wild type Dm7, whereas transformant under-producing Dd-aco (ACO-RNAi) produced a smaller amount of ethylene than Dm7. ACOOE cells formed macrocysts, while ACO-RNAi cells failed to form macrocysts independently of culture conditions. The volatile substance(s) released from NC4 cells was also shown to induce macrocyst formation in V12M2 without its mating type NC4 cells.The two life cycles, the sorocarp and the macrocyst formation, are regulated by several environmental conditions, such as light and water. Dm7 cells form sorocarps in the light, whereas they form macrocysts in the dark or in the water. These environmental conditions affect the synthesis of chemical regulators within the cells. The two chemicals, ethylene and cAMP, have been demonstrated as chemical regulators for choice of developmental pathways in Dm7.Dictyostelium, it has been supposed that ethylene is synthesized from methionine through S-adenosyl-L-methionine (SAM) and ACC as in the case of higher plants.Dictyostelium also suggested the pathway of ethylene biosynthesis in Dictyostelium as the same as in higher plants. Recently, the existence of ethylene receptor in Dictyostelium was suggested by the use of 1-methylcyclopropene (1-MCP), a specific inhibitor of ethylene action through its specific binding to the ethylene receptor, though the ethylene receptor in Dictyostelium has not been identified yet.Dictyostelium cells must share many cellular aspects with higher plants through the ethylene receptor.Concerning the biosynthesis of ethylene in There are several cellular events occurring in the process of macrocyst formation, such as zygote formation and engulfment of the other cells by the zygote in a cell mass, etc. Which of the cellular events are controlled by ethylene? In order to determine the developmental stages when ethylene is effective to induce macrocyst formation, ethylene gas produced by Dm7 and MF1 cultures was removed by use of charcoal at various times of development. As a result, the developmental fate was shifted from macrocyst to sorocarp formation by removal of ethylene at the early developmental stages. This means that ethylene is necessary for macrocyst formation at the early developmental stages. However, ethylene becomes unnecessary for macrocyst formation at the later developmental stages. Since the percentage of sorcarp formation changes in inverse proportion to the ratio of binucleate cells, the timing when ethylene becomes unnecessary for macrocyst formation is consistent with the appearance of binucleate cells regarded as zygotes. The developmental time acquired non-necessity of ethylene for macrocyst formation was delayed when the cell densities plated were decreased. However, when ethylene was applied in the culture dishes at the beginning of this culture, the time acquired non-necessity of ethylene for macrocyst formation advanced in concert with the advanced and increasing formation of binucleate cells .15 TheseThe close relation between the amount of ethylene produced and zygote formation was also shown clearly using the transformant: ACO-RNAi cells never formed zygotes as was expected .26 In heHow does ethylene lead to cell fusion occurring during zygote formation? To address this question, we attempted to find out what molecules are related to signaling pathways involved in the cell fusion during zygote formation. Two strategies were adopted to find out the molecules. 1) Identification of chemicals involved in the signaling pathways for zygote formation. 2) Identification of genes which are specifically expressed in the process of zygote formation.2+) plays an important role in cell fusion in many organisms.2+ was also proposed to be a critical factor for zygote formation including cell fusion in Dictyostelium. The percentage of zygotes was elevated by the presence of extracellular Ca2+.O-tetradecanoylphorbol-13-acetate (TPA), potent activators of protein kinase C (PKC), have been reported to enhance the formation of zygotes.2+ and PKC would act on the induction of zygote formation. It has been documented that the signaling pathway including Ca2+ and PKC is involved in cell fusion during myogenesis.2+ and PKC-mediated signaling pathway.It is well known that calcium ion  inhibit zygote formation through the signaling pathway triggered by cAMP.zyg1 gene was isolated from Dm7 cells as a novel gene expressed predominantly during macrocyst formation .zyg1 gene began 2 hrs after starvation, reached the maximum level at 8 hrs, and decreased when Dm7 cells were cultured under submerged conditions. This expression pattern is quite similar to the developmental kinetics of zygote formation with about 1 hr precedence. The number of zygote began to increase 5 hrs after starvation, reached the maximum level at 9 hrs and then gradually decreased.zyg1 gene was predicted to be involved in zygote formation. In order to confirm this prediction, the zyg1 gene was introduced into Dm7 cells. The transformant overexpressing the zyg1 gene formed macrocysts on agar even in the light which is the condition favourable to sorocarp formation. In addition, they formed many giant cells besides macrocysts. These results suggested that the zyg1 gene might be involved in the induction of zygote formation. The fact that ZYG1 protein has several predicted phorphorylation sites by PKC raised the possibility that ZYG1 protein itself could be a candidate of the substrate of PKC. In general, activated PKC is known to be translocated to the cell membrane. ZYG1 protein is supposed to be translocated from the cytosol to the cell membrane, provided that ZYG1 protein is phosphorylated by PKC. The signaling pathways involved in zygote formation are summarized in Using differential screening of genes, the zyg1 gene during the development in ACOOE cells overproducing ethylene and ACO-RNAi cells under-producing ethylene was examined and compared. The results obtained showed that Dm7 cells and ACOOE cells exhibited higher levels of zyg1 expression, while ACO-RNAi cells exhibited significantly lower levels of expression, coupling with their failure to form zygotes. The reason why no difference of zyg1 expression between Dm7 and ACOOE cells is detected might be due to developmental conditions. The submerged conditions used for extraction of mRNA are favorable to zygote formation. In fact, both Dm7 and ACOOE cells formed macrocysts under submerged conditions. Importantly, zyg1 expression was decreased by application of AOA, an inhibitor of ethylene biosynthesis, into the culture medium. Taken together these results indicate that ethylene induces zygote formation through an enhanced expression of zyg1Ethylene and ZYG1 have a similar function in zygote formation. In order to know the relation between the two, the expression of Dictyostelium have been clarified at the multicellular, cellular and molecular levels as summarized in The functions of ethylene in


Parvovirus (PV) B19 is the causative agent of the childhood disease erythema infectiosum. An association of PV B19 with chronic arthropathies, sometimes resembling rheumatoid arthritis or juvenile idiopathic arthritis (JIA), has repeatedly been described. Other studies, however, have failed to identify any such relationship. In order to study further whether there is a link between PV B19 and JIA, we determined the prevalence of PV B19 specific IgG antibodies in serum samples from children with rheumatoid diseases and compared it with the prevalence in unaffected children We reasoned that if there is an association between PV B19 and JIA, then the prevalence of PV B19 IgG in the children with JIA should be higher than in the control group. PV B19 IgG status was tested in 406 children with JIA and related diseases, and in 146 children constituting a control group. The percentage of PV B19 IgG positive children was not significantly elevated in the disease subgroups compared with age-matched control groups. In conclusion, our findings do not support the hypothesis that human parvovirus B19 is involved in the pathogenesis of JIA. Parvovirus (PV) B19, the causative agent of the childhood disease erythema infectiosum (fifth disease), was identified in 1975. Since then a large spectrum of diseases caused by or associated with PV B19 has been recognized (for review ). In addAn association of PV B19 with chronic arthropathies, sometimes resembling rheumatoid arthritis or juvenile idiopathic arthritis (JIA), has also been described, but other studies have been unable to corroborate these findings. It therefore remains unclear whether there is an aetiological or pathogenic link between PV B19 and rheumatoid arthritis or JIA (for review ).The term JIA encapsulates a heterogeneous group of rheumatic diseases, which – for most subtypes – is distinct from adult rheumatoid arthritis . ManifesIn order to evaluate further the possible link between PV B19 and JIA, we determined the prevalence of PV B19 specific IgG antibodies in serum samples from children with rheumatic diseases and compared it with the prevalence in unaffected children. Whereas PV B19 IgM is detectable only for about 1 to 3 months after an acute infection, PV B19 IgG persists throughout life. Thus, the presence of PV B19 IgG is a marker of previous exposure to PV B19. We hypothesized that if there is an association between PV B19 and JIA, then the prevalence of PV B19 IgG in children with JIA should be higher than in the control group.The study population consisted of children who were referred to the Section of Paediatric Rheumatology at the University of Würzburg, Germany, between 1988 and 2001. Serum samples for routine laboratory studies were obtained from the patients at the initial visit. No selection of patients was performed. Unused serum was kept frozen at -20°C. Demographic data, clinical diagnosis, and information on PV B19 status were extracted from the records. Stored serum samples of children with unknown PV B19 status were retrospectively tested for PV B19 IgG.In addition, serum samples of 146 children were analyzed as controls. These samples were collected from children referred to the Endocrinological Section of the Children's Hospital and from children referred to the Section of Paediatric Rheumatology who presented with complaints not associated with arthropathy or infections.All patients and control children were of white Caucasian descent. The study was conducted in compliance with the Helsinki Declaration and was approved by the ethics committee at the University of Würzburg. Informed consent was obtained from the parents or legal guardians of the children in the arthritis group for standard of care diagnostic procedures, which included serology on infectious agents associated with arthritis. For the control group, the use of anonymized residual serum samples obtained for routine diagnostic procedures was approved by the ethics committee.PV B19 IgG antibodies were determined using an indirect immunofluorescence assay. Briefly, SF9 insect cells infected with baculovirus recombinant for VP1 of PV B19 were spotted on glass slides, air dried, fixed with cold acetone and stored at -20°C until use. For IgG determination, the fixed cells were incubated for 90 min with a fourfold dilution series of each plasma sample, starting at 1:10. Subsequently, the slides were washed with phosphate-buffered saline and incubated with fluoresceine-conjugated goat-anti-human-IgG  and Evans blue for 30 min. After another washing step, coverslips were mounted for immunofluorescence microscopy. The IgG slides were independently read by two experienced persons. If the results did not match, a third person read the slides and the median value was used for further analysis. Samples with a PV B19 IgG titre above 1:10 were counted as positive.P ≤ 0.05 was considered statistically significant.Fisher's exact test was used to compare the seroprevalence proportions of PV B19 between different groups, and the Mann-Whitney test was used to compare PV B19 IgG titers. Between 1988 and 2001, 658 children were referred to the section for paediatric rheumatology. JIA or related rheumatic diseases were diagnosed in 574 children. In 84 of the children, no rheumatic disease was diagnosed. The PV B19 IgG status had already been determined as part of the routine check up in 304 of the children. Of the remaining 270 children in whom information on PV B19 serostatus was not available, stored serum samples were available for 102. Both the children with documented PV B19 status from the initial diagnosis and children in whom PV B19 status was determined retrospectively were included in the analysis . No significant differences were identified (data not shown).Comparing the seroprevalence of PV B19 IgG between children with JIA and a control group, we found no significant difference in PV B19 seropositivity. Our study was based on the assumption that if there is an association between PV B19 and a substantial proportion of JIA cases, then the prevalence of PV B19 IgG in the children with JIA should be higher than that in the control group, irrespective of time of infection. However, our findings do not support an association between PV B19 and JIA.In general, there are two basic approaches to diagnosing viral infections: application of serological tests for antibody detection and application of direct viral detection methods (in particular polymerase chain reaction). Both approaches have been used to study the potential link between B19 and JIA, but thus far no unequivocal evidence favouring or refuting such an association has been presented.Following a description of a case of juvenile chronic arthritis following acute infection with PV B19 , an obseP = 0.006, by Fisher's exact test), but the authors expressed concern in their discussion that statistical bias from case sampling might have occurred. Furthermore, the number of patients with JRA included in the study was low. In addition, the mean age was high, suggesting blood sampling later in the course of disease.In a study conducted in Japan , adult aIn a study conducted in India , 69 chilP = 0.03). Prospective studies such as this one are important in determining whether there is a potential risk for developing JRA after acute PV B19 infection. Unfortunately, acute PV B19 infections in this study were diagnosed solely using a peptide-based PV B19 IgM enzyme immunoassay. Similar peptide-based assays have been found to yield a considerable number of false-positive results compared with assays based on recombinant antigens expressed in the baculovirus system, especially in the presence of autoantibodies and rheumatoid factor [Oguz and coworkers  used a dd factor -22. ConfIn an Italian study, Angelini and colleagues  comparedIn another study of PV B19 and juvenile arthritis, Lehmann and coworkers  examinedThis issue is also pertinent to several other studies, both in adults and in children, that described detection of PV B19 DNA in synovial fluid, cells, and tissue -32. HoweBecause of its retrospective nature, our study has limitations with respect to case finding and description of clinical manifestations. Not all case patients were tested for PV B19 IgG. However, there is no indication of selection bias caused by the exclusion of children for whom stored serum samples were not available. PV B19 polymerase chain reaction and PV B19 IgM tests were not used in this study because of limitations inherent in the interpretation of positive results obtained with these methods, and because the hypothesis to be tested in this study was based on PV B19 IgG status.In summary, there is no conclusive evidence yet for a pathogenic role of PV B19 in JIA. Analysis of the seroprevalence of anti-PV B19 IgG antibodies in European Caucasian children affected by arthritis did not support the hypothesis that human PV B19 is involved in the pathogenesis of JIA.EOPA, early onset pauciarticular arthritis; JIA, juvenile idiopathic arthritis; JRA, juvenile rheumatoid arthritis; NS, nonstructural protein; PV, parvovirus.The authors declare that they have no competing interests.YSF collected the patient data from the clinical records, performed the PV B19 IgG testing, and analyzed the data. BW and HJG designed and coordinated the study. BW contributed to the PV B19 testing and data analysis, and drafted the manuscript. HJG cared for the patients, participated in the analysis of the clinical data and contributed to the manuscript. All authors read and approved the final version of the manuscript.


We report on two patients with fast-growing recurrences of AF resistant to EBRT who obtained protracted clinical benefits with  It has high affinity for the somatostatin receptor subtype 2 (sst2) and a favourable bio-distribution and safety profile in patients   is a rare fibrous neoplasm with biological behaviour intermediate between that of benign fibrous lesions and malignant fibrosarcomas. The tumour is poorly circumscribed and grows along tissue planes with an infiltrating-like pattern towards mesenchymal tissue. The clinical equivalent to this pathological behaviour is a high tendency to local recurrence. Its management is problematic: adequate surgery is the first-choice treatment; however, the lesion tends to recur locally even if surgically removed with pathologically documented free-margin resection. As systemic treatment options, chemotherapy, hormone therapy and NSAIDs have been used with mixed success.The response of AF to external-beam radiation (EBRT) is documented, with a dose–response relation for tumour doses of less than 50 Gy . Two specimens showed immunoreactivity to sst2. Next, we used OctreoScan in six other patients with AF to test the expression of sst2 receptors on the neoplasm.90Y-DOTATOC. We explained to these two patients the potential risks and benefits of this experimental treatment, which had been approved by our Institute's ethical review board. They gave their written consent. An ongoing phase I–II study using 90Y-DOTATOC in patients with solid tumours  and were hence eligible for treatment with 2 receptors, as documented by OctreoScan, and documented residual disease or recurrence after conventional treatment. DOTATOC was synthesised at the Division of Radiological Chemistry, University Hospital, Basel using a published procedure  which has lasted for more than 37 months , there were four relapses in the following 4 years. The relapses were removed surgically and postoperative external-beam reirradiation (35 Gy) was given after the third operation.90Y-DOTATOC in three cycles over 4 months. There was a disease stabilisation on MRI according to WHO criteria after the first cycle (3.26 GBq) and disease has remained stable for more than 19 months.For the fifth relapse, characterised by rapid growth, conservative surgery and radiotherapy were no longer possible, and as an alternative to amputation we administered a cumulative activity of 8.18 GBq of 90Y-DOTATOC was very well tolerated in these patients, and neither acute nor late toxicity was observed.Patients with recurrent AF after full exploitation of the standard therapy modalities such as surgery and EBRT are lacking other local therapeutic options. In this communication, we report on two patients with fast-growing recurrence of AF that appeared to be resistant to appropriate EBRT.90Y-DOTATOC in the absence of acute and late toxicity. The excellent tolerability of this experimental treatment is consistent with the results of our phase I–II study . In this study, toxicity due to 90Y-DOTATOC, which was mainly haematological, occurred in a minority of patients receiving high activities per cycle  and tended to reverse within 4 weeks. Neither permanent renal toxicity nor other forms of toxicity were observed.Both patients obtained a protracted clinical benefit with a peptide receptor radiotherapy with 90Y-DOTATOC can provide clinical benefits in patients with AF, and the benefit obtained supports further investigation in this setting. An OctreoScan should first be performed on a candidate patient with AF and if adequate tumour uptake is observed, 90Y-DOTATOC could be considered as a further treatment option to increase the chance of obtaining disease control.To our knowledge, this is the first report showing that


The interpretation of urothelial atypia in a setting of chronic inflammation and reactive changes can prove difficult with small biopsies. Limited recuts lessen the efficacy of ancillary studies such as CK20, P53 and CD44 in these instances.To evaluate a triple-immunostain with the assistance of multispectral microscopy.Fifty-three bladder biopsies with previous diagnosis of benign/reactive, dysplastic, carcinoma in situ or carcinoma were prepared using a tripleimmunostain cocktail consisting of CK20, P53 and CD44. Three control stains were used for the purpose of creating a spectral library for the Nuance CRI Flex microscopy system. All specimens were interpreted by light microscopy, processed with the Nuance 2.71 software, and CK20 and P53 were scored blinded to the case diagnoses. CD44 was not scored as it proved difficult to interpret in many cases.The results demonstrated that it was possible to separate CK20, P53 and the counterstain that were co-localized in the biopsies. Separation of the stains demonstrated a correlation of p53 and CK20 dual expression in biopsies diagnosed as carcinoma. Low or undetectable levels of expression were seen in biopsies later diagnosed as reactive or benign.The combination of multispectral microscopy and multiple immunostain cocktails form a powerful and useful tool for the interpretation of small biopsies with faint or difficult to interpret staining and for cases with limited material such as small-bladder biopsies. The advantages of immunostains were apparent in 1941 when Albert Coons first described their use.[Multispectral imaging is a technique that captures image information across a broader range of the electromagnetic spectrum than the human eye and traditional RGB cameras. It permits the analysis of tissue with greater spectral densities and resolution of varied overlapping chromogens. There are applications in nearly every subspecialty area of surgical pathology for which using multiple antibodies on the same section would be of great value. This is doubly important in the evaluation of small biopsies where the preservation of scant tissue is sometimes essential. In these instances cutting additional tissue levels carries the risk that the area of interest will no longer be present on the tissue to be stained. The interpretation of small, inflamed or reactive needle-core urothelial biopsies with CK20, P53 and CD44 is one such setting.Our aim is to evaluate a triple-immunostain consisting of CK20, P53 and CD44 with the assistance of multispectral microscopy in order to, in a single slide, evaluate their simultaneous expression and location in urothelium, and, at the same time, diminish the risk of losing the area of interest in further recut sections.After institutional board approval, 53 formalin-fixed and paraffin-embedded bladder biopsies with previous diagnoses of benign/reactive, dysplastic, carcinoma in situ or carcinoma (approximately 39 cases spanning 1998-2006) were retrieved. Four micrometer sections from each were obtained and incubated with a triple-immunostain cocktail composed of CK20, P53 and CD44 antibodies. The CK20 , P53 (DAKO - CM1) and mouse monoclonal CD44  antibodies were added in 1:100, 1:500 and 1:600 concentrations, respectively. Van Gogh yellow diluent Cat# PD902L was added as a diluent and Harris hematoxylin as the counterstain. Biocare Medical chromogens Cardassian DAB Brown Cat#DBC959H, Vulcan Fast Red Cat#FR803S and Bajoran Purple Cat#BJP811L were utilized for the CK20, P53 and CD44 antibodies, respectively.From the control stains a spectral library was created for the Nuance CRI Flex microscopy system . This spectral library is seen in The results, shown in For cases of carcinoma in situ or invasive disease CK20 was expressed in the upper 2/3 or full thickness of the urothelium in 19/26 (73%) of cases while p53 was moderate or heavy in 19/26 cases (73%). As the number of immunostains increase on a slide, particularly when by design they overlap, co-localization and accurate interpretation becomes more difficult. From a laboratory perspective, these “cocktails” can be difficult to design given a limited number of wavelength options, variable staining intensity and inevitable background staining. These limitations in combination make interpretation of such slides difficult and time consuming with the naked eye. Multispectral image analysis is one purported solution for this problem that has been recently studied in fine needle aspiration of the thyroid and examThe staining results presented here are consistent with past studies at the University of Pittsburgh Medical Center performed with an equivalent cocktail. They sup5et al.[As this study was a pilot, there are a number of important limitations worthy of discussion. CD44 expression was poor in the cocktail batch; an unanticipated result given the success with it in previous studies. In selected cases, bright-field analysis was able to compensate to a sufficient extent to reliably discriminate the three stains despite the spectral similarity between the CD44 and P53 chromogens. This was the exception rather than the norm, and therefore, felt to represent anecdotal evidence of Nuance’s ability to manipulate 3 stains under bright-field examination, but requires further scrutiny. Even with appropriate staining, the spectral signatures of the chromogens used with the CD44 and P53 antibodies were overly broad with considerable overlap. Quantum dots have been shown to provide much narrower spectral signatures and would make an interesting comparison. Further investigation is also needed to compare the results from Nuance’s multispectral camera to a “spectral unmixing” RGB method, as described by Ruifrok et al.Lastly, the value of compressing multiple slides into a single tissue section was of limited benefit. With rare exception, extra blank slides cut with the first facing of the block would have sufficed. However, one situation where this value may be realized is an unexpected finding on a cytologically obtained cell block, a setting that invites further investigation.In conclusion, the combination of multispectral microscopy and multiple immunostain cocktails form a promising tool for the interpretation of cases with limited material such as urothelial needle core biopsies. This is true moreover for applications that favor immunohistochemical stains without the need for fluorescent markers or the expense of confocal microscopy. The fields of cytopathology, hematopathology and surgical pathology will serve as prominent subjects for further experimentation in the future.


Chlamydia trachomatis and is known to vary in size between 165 and 237 amino acids, which is caused by different numbers of lysine-rich pentamers. A more complex structure was seen in this study when sequences from 378 specimens covering the hctB gene, which encodes Hc2, were compared.The histone-like protein Hc2 binds DNA in Bordetella but was now also found in other genera, including Burkholderia, Herminiimonas, Minibacterium and Ralstonia.This study shows that the size variation is due to different numbers of 36-amino acid long repetitive elements built up of five pentamers and one hexamer. Deletions and amino acid substitutions result in 14 variants of repetitive elements and these elements are combined into 22 configurations. A protein with similar structure has been described in hctB variants that formed four clades in phylogenetic analysis. Strains causing the eye disease trachoma and strains causing invasive lymphogranuloma venereum infections formed separate clades, while strains from urogenital infections were more heterogeneous. Three cases of recombination were identified. The size variation of Hc2 has previously been attributed to deletions of pentamers but we show that the structure is more complex with both duplication and deletions of 36-amino acid long elements.Sequence determination resulted in 41 Chlamydiacae. The high sequence variation in the corresponding hctB gene enables phylogenetic analysis and provides a suitable target for the genotyping of C. trachomatis.The polymorphisms in Hc2 need to be further investigated in experimental studies since DNA binding is essential for the unique biphasic life cycle of the  Chlamydia trachomatis is an intracellular bacterium that can multiply only within a host cell. Reticulate bodies (RBs) are the replicating form of Chlamydia bacteria that transform into infectious elementary bodies (EBs) prior to cell lysis. The transformation from a fragile RB to a robust EB is dramatic: the size reduces from 1 μm to 0.3 μm, the dispersed chromatin aggregates into a condensed nucleoid and metabolism ceases.C. trachomatis is classified into 19 serotypes (A-L3) based on the major outer membrane protein (MOMP) where A-C cause trachoma, D-K cause urogenital infections and L1-L3 cause lymphogranuloma venereum (LGV).hctA and hctB genes that are expressed late in the life cycle when RBs convert to EBs [hctA gene has been inserted into Escherichia coli and the expressed Hc1 was shown to induce a compaction of chromatin into a spherical condensed nucleoid [Hc1 and Hc2 are DNA-binding proteins, homologous to eukaryotic histone H1 [t to EBs . The hctnucleoid . Hc2 alsnucleoid ,6, indicChlamydiaceae because the hctB gene has been found in all available genome sequences of this family, Based on Southwestern blot analysis, Hc2 has previously been reported to be absent or present in reduced amounts in Chlamydophila psittaci strain Mn [hctB gene has been found in C. psittaci strain 6BC by whole genome sequencing .The Hc1 protein has two domains: the conserved N-terminus , which mtrain Mn . HoweverhctB gene encoding Hc2 is one of the targets in a newly developed multilocus sequence typing (MLST) system for C. trachomatis [hctB sequences are stored in a publically-accessible MLST database [The chomatis . Studieschomatis  and othedatabase .hctB sequences from many C. trachomatis specimens it was clear that the size variation was more complex than could be attributed to simple deletions of a pentamer as previously described. In this study we found elements of 108 bp that are deleted and duplicated within the hctB gene without a premature stop codon or loss of the reading frame. We have created a nomenclature to characterise the variation in numbers and type of these elements observed in 378 clinically derived and reference specimens of C. trachomatis.When comparing hctB gene variants were found among 378 sequences in the MLST database, with the highest level of variation occurring in a region encoding consecutive amino acid pentamers. The pentamers have two positively charged residues (arginine and lysine) and three other residues that are mainly alanine, but also valine, threonine and proline  based on the protein sequence Figure . The 1, hctB gene for 17 strains representing different types of Hc2. Only three variable positions were found. Two substitutions, of which one is silent, separate the LGV serovars from the others and one silent substitution is unique for the D, G, H, J and K serovars.The amino acid sequence outside the variable region is highly conserved with no insertions or deletions. The beginning of the gene encodes 24 amino acids with two substitutions; one of these substitutions is restricted to the B , D, G, H, I, Ia, J and K serovars while the other is found in some trachoma strains. The last 69 amino acids of Hc2 downstream of the variable region are therefore partly excluded in MLST typing analysis. The only differences in sequence found in the 87 bp obtained with MLST sequencing are two substitutions that both cause a change in amino acid. One substitution was unique for the D, G, H, J and K serovars and one was found only in a trachoma strain. Additional sequencing was done in order to cover the last 120 bp of the hctB . The sequences with a possible recombined origin are 36_J, 37_J (same event), 12_DHJK and 30_G. Removing these sequences from the dataset before Bayesian analysis gave the same overall topology (data not shown), but with an increased number of clades with significant support.C. trachomatis, one copy resulting in the 1, 2, 6 and 7 group and the other in the group comprising the element types 3-5 and 8-14. Because the 1, 2, 6 and 7 elements are always found one per sequence and first in order, the structure can be described as 1 + 1-3 elements rather than 2-4. Mapping this pattern on the hctB phylogeny, the first element  appeared to have evolved by substitutions and deletions only. The 2 element for example can have evolved through a series of nucleotide substitutions, or by deletion of the end of a 1 element and the beginning of a 4 element. The remaining elements (3-5 and 8-14 super group) appear to have a much higher rate of duplications and extinction of entire elements. Thus in a duplication of a 5b element one copy gave rise to the 3 group lineage and the other copy to 5a and subsequently to the 4 group lineage of elements, with later duplications and extinctions within both these lineages.The phylogenetic analysis of the repeat element types Figure  indicateC. trachomatis specimens. Sequence comparisons show that Hc2 is a highly structured protein with consecutive pentamers but also with repetitions of larger elements built up by six pentamers and one hexamer. These repeated elements were found in 14 amino acid variants combined differently resulting in 20 configurations and 11 length variants of Hc2.Hc2 displays considerable diversity in length and in sequence when comparing 378 C. trachomatis because there are specimens with different configurations of repetitive elements but with identical ompA genotype and MLST profile. The diversity generated by several deletions and duplications while the flanking regions remain intact suggests that the Hc2 protein is vital for Chlamydia, and that the number of repetitions in the DNA-binding region has an important role for the organism. It is difficult to link the length of Hc2 to particular characteristics because many specimens in the MLST database lack additional information such as clinical manifestations and phenotypic differences. This needs further exploration. However, the size variation enabled by rearrangement of the repeated elements might be beneficial for the regulation of DNA condensation and gene expression during the transition from RB to EB.The rearrangement of repetitive elements appears to be continuous in ompA gene [porB gene gives a different classification in which the ocular and LGV strains are in separate clades [hctB, where the ocular strains are found in clade I and the LGV strains in clade V  genotyping system [C. pneumoniae are all identical in hctB. C. pneumoniae is difficult to differentiate with highly discriminatory methods  [C. trachomatis when using AFLP [The g system . In contnalysis)  and is ming AFLP  or MLST ing AFLP .Bordetella (5 sequences), Burkholderia (31 sequences), Herminiimonas (1 sequence), Minibacterium (1 sequence) and Ralstonia (4 sequences). These proteins have a similar amino acid composition and similar pentamers, resulting in a distribution of positively charged residues almost identical to Hc2  among 31 sequences from nine species. Longer repeats and several different kinds of repeats in the same protein were found in Burkholderia ambifaria, Burkholderia cenocepacia, Burkholderia pseudomallei, Burkholderia vietnamensis and Burkholderia multivorans. On the other hand, short consecutive repeats of only a pentamer were repeated seven and nine times in Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. The Hc2-like proteins in Bordetella petrii and Burkholderia phymatum have no repeats. The protein most similar to Hc2 in C. trachomatis was found in Herminiimonas arsenicoxydans . The repetitive elements were aligned based on homology according to neighbour-joining phylogenetic analysis of the different types of repeat element. Obtained sequence variants were submitted to GenBank and the accession numbers are listed in additional file Chlamydiales and Hc2-like proteins in other genera are listed in additional file The MLST database  containeescribed  but withhttp://www.emboss.org).Repetitive amino acid elements were found with Dottup plots using a word size of 20 and Pepinfo was used to create plots that show the charge distribution. Both Dottup and Pepinfo are part of EMBOSS . The posterior probabilities were then summarized as a consensus tree with MrBayes.MrBayes uses a Metropolis-coupled Markov chain Monte Carlo method to compute the posterior probabilities for the clades. This algorithm has no defined stop condition, but runs for a number of generations and must be monitored for convergence, and thus completion, of the algorithm. The convergence was assessed by monitoring the continuous-valued parameters using the software Tracer 1.4 , resultiThirdly, the consensus tree was rooted by paralog rooting  based onLastly, to check for conflicting signals and possible patterns of recombination, a recombination network of the sequences was computed using SplitsTree 4.10 .MK and BH designed the study and MK wrote the manuscript draft. MK and AN performed the sequence determination, analysed obtained sequences and identified the repetitive elements. MT performed the phylogenetic analyses. EBR analyzed protein strucures and together with MK found Hc2-like proteins in other genera. SB contributed intellectually since he has studied the Hc2 protein in the past. All authors participated in the writing process.Appendix 1. List of the 378 sequences in the MLST database included in this study.Click here for fileAppendix 2. Sequence variants of the MLST target that include hctB in Chlamydia trachomatis with corresponding accession number. Each sequence variant is named after the allele number and the serotypes in which that variant has been found.Click here for fileAppendix 3. Hc2 amino acid sequences in Chlamydiales and Hc2-like sequences in other genera.Click here for file