We recently characterized HAmo SINE and its partner LINE in silver carp and bighead carp based on hybridization capture of repetitive elements from digested genomic DNA in solution using a bead-probe . To reve4 to 106 in cyprinid genomes by quantitative RT-PCR. Over one hundred type IV members were identified and characterized in the primitive cyprinid Danio rerio genome but only tens of sequences were found to be similar with type I, II and III since the type IV was the oldest subfamily and its members dispersed in almost all investigated cyprinid fishes. For determining the taxonomic distribution of HAmo SINE, inter-primer SINE PCR was conducted in other non-cyprinid fishes, the results shows that HAmo SINE- related sequences may disperse in other families of order Cypriniforms but absent in other orders of bony fishes: Siluriformes, Polypteriformes, Lepidosteiformes, Acipenseriformes and Osteoglossiforms.Sixty-seven full-size and 125 internal-SINE sequences  from 17 species of the family Cyprinidae were aligned as well as 14 new isolated HAmoL2 sequences. Four subfamilies , which were divided based on diagnostic nucleotides in the tRNA-unrelated region, expanded preferentially within a certain lineage or within the whole family of Cyprinidae as multiple active source genes. The copy numbers of HAmo SINEs were estimated to vary from 10Depending on HAmo LINE2, multiple source genes (subfamilies) of HAmo SINE actively expanded and underwent retroposition in a certain lineage or within the whole family of Cyprinidae. From this perspective, HAmo SINE should provide useful phylogenetic makers for future analyses of the evolutionary relationships among species in the family Cyprinidae. Retrotransposons are widely distributed among eukaryotic genomes and occupy a substantial fraction of genome. These repeats increase in number by retroposition, which involves transcription of their genomic copies followed by reverse transcription of an RNA intermediate and results in cDNAs reintegration into the genome host -4. RetroBoth LINEs and SINEs become amplified in genomes during evolution, but while LINEs encode the enzymes required for their retrotransposition, SINEs presumably borrow these enzymes from other sources, most likely from LINEs ,7. Resea5 copies per genome. Many SINEs can be categorized into families based on sequence similarity and into subfamilies based on the presence of diagnostic nucleotides and/or deletions. An active member of a subfamily yields new copies through retroposition. After their dispersion within the genome, individual nonactive members of a subfamily accumulate mutations randomly. Thus, the average sequence divergence of members of a subfamily can be used to roughly estimate the age of the subfamily [SINEs are short (approximately 80 to 400 bp) repetitive elements that often are present at more than 10ubfamily .A typical SINE is composed of three parts: the tRNA-related region, the tRNA-unrelated region and the LINE-derived region with a polyA or short duplication tail. The existence of RNA Pol III promoters in the tRNA-related region ensures the transcriptional activity of SINEs, whereas the LINE-derived region allows the SINE to utilize the LINEs's enzymes for retrotransposition. The central region (the tRNA-unrelated region) between the tRNA- and LINE-derived regions is quite different and is not strongly conserved between different families, although two conserved central regions have been reported for two SINE families (V-SINE and Core-SINE) ,14.To reveal the species distribution and evolutionary history of SINEs and LINEs in more cyprinid genomes, we performed a multi-species search for HAmo SINE and its partner LINE in almost all subfamilies of Cyprinidae, as well as in distantly related fishes  using the bead-probe capture and internal-SINE-primer polymerase chain reaction (PCR) techniques. The detailed sequence alignment and nucleotide diversity estimation of HAmo SINE characterized to date enabled us to understand amplification and evolution of HAmo SINE in cyprinid fishes.Table A rapid bead-probe capture technique based on magnetic separation was used to isolate full-size of HAmo SINE copies from unknown genomes as described previously . The cloInternal-primer-SINE PCR was conducted to amplify the internal region corresponding to residues 18-144 of the whole HAmo SINE consensus sequences (residues 1-150) using a pair of primers . The PCR was run in a total volume of 20 μl including 200 ng DNA template with 25 cycles of 95°C for 40 s, 62°C for 40 s, and 72°C for 40 s. The internal-primer-SINE-PCR was conducted in the selected 17 species of Cyprinidae to detect many individual SINE copies. Finally, about 10 clones were characterized and sequenced for each species.Leuciscus tumensis based on the internal-primer-SINE PCR.Many HAmo sequences  were isolated and characterized from distinct cyprinids fishes. Each locus was named after the number of the clone and the name of the species from which it was isolated. In the middle, F or I was used to represent the internal SINE sequence or full-size sequence obtained from the bead-probe capture (F) or internal-primer-SINE PCR (I), respectively. For example, Ltu_I_4 means that the number of the clone was 4 and it was isolated from Plasmid Hmo41_It and Genomic DNA were prepared as the standard and samples for Real-Time PCR, respectively Table . Their cPhylogenetic analysis was performed using Bayesian inference within the program MrBayes 3.1.1 ,18. The lys-related promoter region at the 5'end, a unique family-specific region, and an end with a HAmo-LINE2-derived 3' terminus preceding the TAAATG short tandem repeat. Remarkably, HAmo SINE seems to be widely distributed in cyprinid genomes and multiple subfamilies may exist based on preliminary observation of their high diversity.Figure Internal-primer-SINE PCR which amplified fragments corresponding to residues 18-144 of the HAmo SINE consensus sequence (residues 1-150) were conducted. The result showed that HAmo SINE was present in all 17 representatives of the family Cyprinidae tested in this study. To unambiguously confirm them and obtain enough HAmo SINE sequences to observe their diversities, we cloned the positive fragments and sequenced about 10 positive clones for each species. Moreover, 125 internal sequences corresponding to different individual HAmo-SINE copies from the 17 cyprinid genomes were isolated and characterized.Sixty-seven full-size sequences and 125 internal-SINE sequences  [Figure The four subfamilies can be distinguished from each other mainly by their different tRNA-unrelated regions. The different patterns of insertion and duplication in different subfamilies constitute the most diagnostic nucleotides that lead to division of the family into different subfamilies. Type I has a variable polyA (1-14) insertions and a TATAA repeat two to three times. Type II has two variable polyA insertions whereas type III has a TAAATG sequence that repeats two times.Many HAmo LINE2 clones were obtained simultaneously when isolating HAmo SINEs Figure  because 4 to 106. In other words, HAmo SINE may constitute 0.1-10% of cyprinid genomes (about 109 bp). Considering the possibility of mismatch between primers with more divergent HAmo SINE sequences, the results of quantitative RT-PCR were minimal estimates of HAmo SINE copy numbers in respective genomes.To estimate the number of copies of HAmo SINE in each species, the pair of primers used in internal-primer-SINE-PCR was also used to amplify the HAmo SINE sequences in genomic DNA (sample) and in the plasmid Hmo41_It (standard) using RT-PCR. We used two diluted genomic DNA as tested samples to perform PCR reaction. The final estimations of copy numbers using different concentrations of the genomic DNA template were very close, suggesting that the results of the experiment were stable and efficient. Table Danio rerio was a cyprinid fish belonging to Danioninae, Cyprinidae. In previous research, the trees resulting from cyprinid phylogenetic analyses of the RAG2 gene place the Danioninae as the basal-most group in the family and divide the remaining cyprinids into two divergent subfamilies Cyprininae and Leuciscinae . In thisFor investigating the HAmo SINE in zebrafish, the consensus sequences of four subfamilies  were used as queries to blastn Danio rerio database in NCBI website. Because the primer regions of these consensus sequences are identical and uninformative, we eliminated these regions from the whole consensus sequences, as shown in Figure Moreover, among our isolated HAmo internal sequences in 17 cyprinid fishes, there are tens of HAmo sequences that have more divergent central region with each other as well as with the type I, II, III and IV Figure . We coul4-106.In this work, we isolated and characterized many HAmo SINEs from 17 species of the Cyprinidae family. The results show that the HAmo SINE is widespread in cyprinid genomes and that copy numbers are about 10Based on the rag2 gene, we constructed the phylogenetic relationships among the 17 species studied in order to map the information obtained from isolated clones in each species Figure . The ratFrom the mapping results, type I seems to have expanded actively in the Leuciscinae clade and to have undergone retroposition among populations of silver carp and bighead carp. However, type I seems not to have flourished in the Cyprininae clade, as there are only two clones found in two species. Thus, we deduced that the master (source) gene of type I was first generated in a common ancestor of the family Cyprinidae, and then this gene was vertically inherited during evolution of the cyprinid lineage. A highly dominant source gene for the type I subfamily may have been newly created by retroposition and actively amplified in the Leuciscinae clade. This scenario indicates that the actual sequence of this subfamily might not be the cause of the high retropositional efficiency of SINEs. A local environment such as the site of retroposition, RNA secondary structure, and promoter activity might be crucial for the establishment of a highly dominant source gene during evolution .The above analysis indicates that multiple source genes (subfamilies) actively amplified within a certain lineage or within the whole family of Cyprinidae. Therefore, the changes (diagnostic nucleotides) that occurred in the tRNA-unrelated region may not affect the efficiency of retroposition of different actively dominant source genes (subfamilies). The high efficiency and successful proliferation of HAmo SINEs might have occurred for two reasons. First, HAmo SINEs retain the overall secondary structure and conserved the A and B box in the tRNA-related region, which ensures the RNA pol III recognition and transcriptional activity of SINEs. Second, the 3' tail of HAmo SINEs and HAmo LINE2 is almost identical in primary sequence and secondary structures, which allows the former to utilize the LINE2 enzymatic machinery.To determine the species distribution of HAmo SINE, Inter-primer SINE PCR was also conducted in non-cyprinid fishes. Four different ten-fold DNA dosage: 0.1 ng, 1 ng, 10 ng, 100 ng were used for each species. Firstly, for 17 cyprinid fishes, visible PCR band could be observed when 0.1 ng DNA was used as template in PCR Figure . SecondlIn Siluriformes, two species were used: Gagata dolichonema (Sisoridae), Pseudobagrus truncatus (Bagridae). Some PCR products were generated only when 100 ng DNA template were used Figure . We sequThus, the PCR analysis has demonstrated that the genomes of representatives of families Catostomidae, Cobitidae, Gyrinocheilidae and Homalopteridae may contain HAmo related SINE elements whereas the genomes of other order Siluriformes, Polypteriformes, Lepidosteiformes, Acipenseriformes, Osteoglossiforms may do not carry this retroposon.In conclusion, by searching for HAmo SINEs from different taxa within the Cyprinidae, we found that HAmo SINEs were widespread in cyprinid genomes. The hundreds of SINE sequences found in our study revealed that multiple source subfamilies actively expanded and underwent retroposition within a certain lineage or within the whole family of Cyprinidae. Thus, HAmo SINE should be a useful phylogenetic marker for future analyses of the evolutionary relationships among species in the Cyprinidae family.SINE: short interspersed repetitive elements; LINE: long interspersed repetitive elements; qRT-PCR: Quantitative Real Time-PCR; RT: reverse transcriptase.SH and CT conceived and designed the experiments, CT and XG performed the experiments and analyzed the data, CT and SH wrote the paper. All authors read and approved the final manuscript.Type IV of HAmo SINE in zebrafish. The alignment of type IV sequences in zebrafish with the consensus sequence of type IV in this paper was provided.Click here for fileType U of HAmo SINE in cyprinid fishes. Among our isolated HAmo internal sequences in 17 cyprinid fishes, there are tens of HAmo sequences that have more divergent central region with each other as well as with the type I, II, III and IV. We defined them as type U (unidentified) because we couldn't divide them into specific subfamilies now.Click here for fileHAmo SINE in zebrafish and other families of order Cypriniform isolated from inter-primer SINE PCR products. HAmo SINE and HAmo SINE-related sequences by cloning the inter-primer SINE PCR products were isolated in zebrafish and some species from families of order Cypriniform. Dre stands for Danio rerio(Cyprinidae); Gay stands for Gyrinocheilus aymonieri (Gyrinocheilidae); Hab stands for Hemimyzon abbreviata ; Mas stands for Myxocyprinus asiaticus (Catostomidae).Click here for file


Numerous non-surgical interventions have been proposed for this disorder, however there is limited evidence for their efficacy. Intra-articular injections of hyaluronan have shown beneficial effects in case-series and clinical trials for the treatment of osteoarthritis of the first metatarsophalangeal joint. However, no study has evaluated the efficacy of this form of treatment using a randomised placebo controlled trial. This article describes the design of a randomised placebo controlled trial to evaluate the efficacy of intra-articular hyaluronan (Synvisc®) to reduce pain and improve function in people with hallux limitus.Osteoarthritis of the first metatarsophalangeal joint (MPJ) of the foot, termed One hundred and fifty community-dwelling men and women aged 18 years and over with hallux limitus (who satisfy inclusion and exclusion criteria) will be recruited.®) or sterile saline (placebo) into the first MPJ. The injections will be performed by an interventional radiologist using fluoroscopy to ensure accurate deposition of the hyaluronan in the joint. Participants will be given the option of a second and final intra-articular injection  either 1 or 3 months post-treatment if there is no improvement in pain and the participant has not experienced severe adverse effects after the first injection. The primary outcome measures will be the pain and function subscales of the Foot Health Status Questionnaire. The secondary outcome measures will be pain at the first MPJ , stiffness at the first MPJ, passive non-weightbearing dorsiflexion of the first MPJ, plantar flexion strength of the toe-flexors of the hallux, global satisfaction with the treatment, health-related quality of life (assessed using the Short-Form-36 version two questionnaire), magnitude of symptom change, use of pain-relieving medication and changes in dynamic plantar pressure distribution (maximum force and peak pressure) during walking. Data will be collected at baseline, then 1, 3 and 6 months post-treatment. Data will be analysed using the intention to treat principle.Participants will be randomised, using a computer-generated random number sequence, to receive a single intra-articular injection of up to 1 ml hyaluronan (Synvisc®) for the treatment of osteoarthritis of the first MPJ . The study has been pragmatically designed to ensure that the study findings can be implemented into clinical practice if this form of treatment is found to be an effective treatment strategy.This study is the first randomised placebo controlled trial to evaluate the efficacy of intra-articular hyaluronan (SynviscAustralian New Zealand Clinical Trials Registry: ACTRN12607000654459 Th. Th30]. ® plantar pressure measurement device [® plantar pressure measurement device   Figure . This syd Figure . The tes® system . The two-step gait initiation protocol will be used to obtain foot pressure data, as it requires fewer trials than the mid-gait protocol and has similar re-test reliability [® software (version 5.24) will be used to construct individual "masks" to determine maximum force (kg) and peak pressure (kg/cm2) under seven regions of the foot: hallux, lesser toes, 1st MPJ, 2nd MPJ, 3rd to 5th MPJs, midfoot and heel  scale. The five point-Likert scale will ask "How satisfied are you with the treatment you received for your big-toe joint pain?", and will have the following five responses: "Dissatisfied", "Only moderately satisfied", "Fairly satisfied", "Clearly satisfied" and "Very satisfied". The dichotomous scale of satisfaction will be answered as "Yes"' or "No" in response to the question: "Would you recommend the treatment that you received to someone else with big-toe joint pain".Physical health and Mental health. The Short Form-36 (SF-36) has been extensively validated and is one of the most widely used instruments to measure health status. The SF-36 shows content, concurrent, criterion, construct, and predictive evidence of validity. The reliability of the eight concepts and two summary measures has been assessed using both internal consistency and test-retest methods. Reliability statistics have exceeded 0.80 [The Short-Form-36 (version two) (SF-36) questionnaire will be used to assess health related quality of life. The SF-36 is a 36 question survey that measures eight health concepts most affected by disease and treatment. The eight health concepts can then be used to form two summary measures: ded 0.80 -37.Self-reported magnitude of symptom change will be measured using a 15-point Likert scale. The scale will ask participants "how much have your symptoms in your big-toe joint have changed from the beginning of the study to now?". The fifteen responses will range from "A very great deal better" to "A very great deal worse".The number of participants who consumed rescue medication  and mean consumption of rescue medication to relieve pain at the first MPJ  and types of adverse events (including adverse drug reactions) in each treatment group during the trial will be recorded using a questionnaire that participants will complete during the follow-up appointments at 1, 3 and 6 months post-treatment . To clasa priori power analysis using the primary outcome measure of the pain domain of the FHSQ [The sample size for the study has been pre-specified using an the FHSQ . One hunthe FHSQ  and a stthe FHSQ . This cathe FHSQ . HoweverStatistical analysis will be undertaken using SPSS version 14.0  and STATA 8  statistical software. All analyses will be conducted on an intention-to-treat principle using all randomised participants -47. MissDemographic characteristics  will be determined for the baseline visit for each treatment group. Summary statistics will be calculated for duration of symptoms, side affected , grade of OA at the first MPJ  as well d  or odds ratios  as appropriate.Analyses will be conducted on 1, 3 and 6 month outcome measures. The continuously-scored outcome measures at 1, 3 and 6 months will be compared using analysis of covariance with baseline scores and intervention group entered as independent variables ,50. The ® or sterile saline according to the treatment group they are in) on days 30 or 90 respectively, will also be analysed as described above. These analyses will be classified as secondary outcomes.The outcome measurements obtained at 7 or 9 months for participants that receive a second and final intra-articular injection (of Synvisc®) to reduce pain and improve function in people with OA of the first MPJ . Two studies have previously investigated the efficacy of intra-articular hyaluronan for the treatment of first MPJ OA [This study is a randomised placebo controlled trial designed to investigate the efficacy of intra-articular hyaluronan  on days 30 or 90 if there is no improvement in their symptoms. Although this has the potential to complicate the interpretation of the results of the study, this protocol was included as it is likely to be more reflective of clinical practice [Participants will be given the option of a second and final intra-articular injection (of Synviscpractice , and thiIn summary, this project is the first randomised controlled trial to be conducted to evaluate the efficacy of intra-articular hyaluronan for reducing pain and improving function in people with hallux limitus. The study protocol, including interventions, have been pragmatically designed to ensure that the study findings are generaliseable to clinical practice. Recruitment for the study will commence in June 2008, and we expect final results to be available in mid-2010.Journal of Foot and Ankle Research. It is journal policy that editors are removed from the peer review and editorial decision making processes for papers they have co-authored.HBM and KBL are Editor-in-Chief and Deputy Editor-in-Chief, respectively, of SEM, HBM, KBL and CJH conceived the idea and obtained funding for the study. SEM, HBM, KBL, AEZ and JDL designed the trial protocol. SEM, HBM, KBL and GVZ drafted the manuscript. All authors have read and approved the final manuscript.


Previous studies showed that gene hybrid is one of the principal processes for generating new genes. Although some gene hybrid events have been reported to be inter- or intra-species, there lacks a well-organized method for large scale detection of the events with multiple components. Hence in this study, we focus on building up an efficient method for exploring all candidates of gene hybrid events in human genome and provide useful results for further study.N-hybrid events  in human genome. The results reveal that there are many convoluted N-hybrid events with multiple components (N > 2) and that the most complicated N-hybrid genes detected in human by TCA are composed of six component regions. Interestingly, our results show that most of the hybrid events belong to the 3-hybrid category. Furthermore, we observe that a single gene might participate in different events. Twelve genes were found to have dual identities contained in different N-hybrid events . This points out that to a certain extent the gene hybrid mechanism has generated new genes during the course of human genome evolutionary history.We have developed a method designated Triad Comparison Algorithm (TCA) to detect all potential An efficient method, TCA, is developed for exploring all candidates of hybrid genes in the human genome and provides useful results for the evolutionary analysis. The advantage of TCA is its power of detecting any kinds of hybrid events in any species with a large genome size. Drosophila melanogaster was originated from retroposition followed by gene fission event [The emergence of new genes is fundamental to the evolution of lineage- or species- specific traits . Duplicaon event .N) of components because they are based on pair-wise sequence comparison. Therefore, their computational complexity will increase exponentially as N increases, leading to a difficulty of the large scale detection, especially for multiple components.Recently, the gene fusion/fission has been demonstrated to largely contribute to the evolution of multi-domain proteins in bacteria . From liN components, where N ≧ 2) with flexible criteria in the human genome. This result reveals that some of the hybrid events are complicated and that some genes seem to undergo such the events multiple times in the human genome.In this study, we have built up an efficient method for exhaustively exploring all candidates of gene hybrid events . An N-hybrid gene is a gene that contains N non-overlapping component regions derived from N different genes . These N component genes are defined as being not alignable with each other. Here, we also term a 2-hybrid event as a "triad", which is the basic unit for identifying all other N-hybrid events (N > 2) in the genome studied. A model of a triad is illustrated in Figure We propose a progressive algorithm, Triad Comparison Algorithm (TCA), to identify . There are 20,181 groups for 38,086 transcript IDs and all isoform groups are different from each other. We used BLAST(2.2.11) program from NCBI to do the pair-wise comparison for all transcript sequences. After collecting the alignment score matrix , we set up filtering criteria  to define the relatedness between gene pairs and used TCA to detect all triads (2-hybrid events). Based on the triads, we can further search N-hybrid events with N > 2. The flowchart of this study is shown in Figure We download the known-Isoforms file HG17) of human genes from UCSC website 7 of humaN-hybrid events in transcription sequence level. The summary of all detected results are shown in Table N-hybrid events , we calculate the length proportion of components to hybrid gene for each N-hybrid event. Because the tendency is similar for all length criteria of components, we present the results with 50 and 100 bp in Table S1 . Table S1 shows that the 3-hybrid event has the smallest proportion, on average, came from the corresponding component genes under all different length criteria. Although each component region contained in 3-hybrid genes usually has longer length than that contained in 2-hybrid genes, but on average the length of 3-hybrid genes is even longer than that of 2-hybrid genes. This conduces that 3-hybrid event has the smaller proportion of component regions than 2-hybrid event. When the length criteria of component regions are larger than 100 nucleotides, only few N-hybrid events with N = 4 and 5 have been found  genes. They are listed in Table S3  proteins, on average, are involved in each fusion event [N-hybrid events are 3-hybrid in the human genome.Remarkably, we detected that the majority of hybrid events are 3-hybrid regardless of the component length criteria used Table , while aormation ,22,23. Fhan 2 ~2.4 proteinon event ,29. If wWe propose a hypothesis that 3-hybrid event is the main composition of gene hybrid mechanism in the human genome. The complete human gene network based on their functional roles is not currently available, yet our preliminary analysis for gene hybrid events still gives some insights on how human genes produced by hybrid mechanism. The study for the demonstration will proceed in the future.N-hybrid events in the human genome. Our result reveals that the hybrid mechanism between genes is an important way to generate new genes in this genome. Furthermore, the results also reveal that one gene could be involved and play opposite roles in different hybrid events. This phenomenon suggests the possibility of multiple occurrences of hybrid mechanism in human genome evolution. It further suggests that the hybrid mechanism is not just an accidental event but an on-going process in the human genome evolution. Another important insight from our results is that the 3-hybrid events may be the basic unit in the complicated hybrid gene network.In this study, we have developed a method, TCA, for the detection of all potential The authors declare that they have no competing interests.YHT conceived the study and prepared the manuscript. YHT and SSW contributed computational concepts and analyzed the data. All authors read and approved the final manuscript.Triad Comparison Algorithm. The detailed description the algorithm and the method of detection for all potential N-hybrid events in human genome.Click here for fileTable S1. The average length of a single component region, N-hybrid gene, and the proportion of all components contained in N-hybrid genes under different length criteria (50 and 100 bp).Click here for fileTable S2. The number of events, N-hybrid genes, and component genes of all N-hybrid events with each component length > 50 nucleotides.Click here for fileTable S3. List of all mixed Rosetta Stone (MRS) genes and some examples of the involved hybrid events with dual identities.Click here for fileN-Hybrid EventsAll . The detailed information of all detected N-hybrid events by using the alignment parameters of E-value < 10-10, identity > 70%, and alignment length > 50 bp between component regions of N-hybrid genes and their corresponding component genes.Click here for fileAll Images. Graph representations of all N-hybrid events in the Additional file Click here for file


MYCN in double minutes (DMs) is a common finding in neuroblastoma (NB). Because DMs lack centromeric sequences it has been unclear how NB cells retain and amplify extrachromosomal MYCN copies during tumour development.Amplification of the oncogene MYCN-carrying DMs in NB cells translocate from the nuclear interior to the periphery of the condensing chromatin at transition from interphase to prophase and are preferentially located adjacent to the telomere repeat sequences of the chromosomes throughout cell division. However, DM segregation was not affected by disruption of the telosome nucleoprotein complex and DMs readily migrated from human to murine chromatin in human/mouse cell hybrids, indicating that they do not bind to specific positional elements in human chromosomes. Scoring DM copy-numbers in ana/telophase cells revealed that DM segregation could be closely approximated by a binomial random distribution. Colony-forming assay demonstrated a strong growth-advantage for NB cells with high DM (MYCN) copy-numbers, compared to NB cells with lower copy-numbers. In fact, the overall distribution of DMs in growing NB cell populations could be readily reproduced by a mathematical model assuming binomial segregation at cell division combined with a proliferative advantage for cells with high DM copy-numbers.We show that MYCN copy-number variability in NB. Our findings also provide a proof-of-principle for oncogene amplification through creation of genetic diversity by random events followed by Darwinian selection.Binomial segregation at cell division explains the high degree of  MYCN, which is observed in ≈30% of NB MYCN is associated with advanced stages of disease and the 3-year event-free survival of tumours with MYCN amplification is <20% ODC, MCM7, and MDR1MYCN, or MYCN copy-number elimination, leads to growth-arrest, senescence and apoptosis of NB cells MYCN amplicons are typically carried either in extrachromosomal double minute (DMs) or in intrachromosomal homogeneously staining regions (HSRs). Under in vitro conditions HSR is the most common manifestation of MYCN amplification, whereas in fresh NB biopsies the amplicons are typically carried by DMs MYCN gene amplification by generating cell populations with heterogeneous DM copy-numbers on which Darwinian principles could be imposed.Neuroblastomas (NB) are tumours of the sympathetic nervous system, occurring predominantly in early childhood and accounting for 8–10% of all paediatric cancers. One of the most important prognostic markers in NB is amplification of the oncogene MYCN amplicons in the two NB cell lines CHP-212 and SK-N-AS were detected by combined fluorescence in situ hybridization (FISH) and beta-tubulin immunofluorescence using epifluorescence microscopy , DMs were distributed evenly in the nucleus. The remaining nuclei either exhibited DMs restricted to the nuclear periphery (10–14%) or a preferential localisation of DMs to nuclear membrane protrusions or micronuclei (<1%). In contrast, prometaphase and metaphase cells (>100 cells/cell line scored) invariably showed DMs only at the periphery of the prometaphase rosette/metaphase plate. At anaphase, the DMs in all three cell lines remained at the periphery of the chromosome poles and were typically (>90% of DMs) located at, or adjacent to, the termini of the chromosomes (>50 cells/cell line scored). This configuration was also observed in telophase cells. DMs that had detached from the chromosomes were not included in the analyses; these consisted of <10% of observed DMs.To locate DMs in relation to chromosomes through the cell cycle, croscopy . As a coIn order to characterize the mitotic distribution of DMs during the cell cycle in three dimensions, we then performed confocal microscopy of mitotic and interphase cells in the three cell lines (>20 mitotic cells and >10 interphase cells scored in each line). DMs were classified as peripherally located if the corresponding FISH signals overlapped with the chromatin border as assessed by DRAQ5 counterstaining . From prMYCN and TTAGGG-sequence probes assessed by epifluorescence microscopy showed that approximately 80% of MYCN signals overlapped with signals from telomeric repeats in mitotic cells from prometaphase through telophase  probes was also performed in LA-N-5 and CHP-212. On average, only 4% and 6% of MYCN signals, respectively, overlapped with centromere signals in mitotic cells and interphase cells .The mitotic location of DMs adjacent to chromosome termini prompted us to investigate whether DMs co-localized with telomeric repeat sequences in LA-N-5 and CHP-212. Co-hybridization with elophase . In contTo validate our fluorescence microscopy data by an additional method, we performed further analysis of DM topography in mitotic CHP-212 cells by combined atomic force microscopy (AFM) and FISH. AFM is a well established technique in material sciences, making it possible to map surface topography with nanometre resolution. Superimposition of FISH images and AFM topography plots made it possible to establish the location of single and clustered DMs with high precision in three dimensions and confirmed the peripheral localisation of DMs at mitosis . ApproxiMYCN or MYC probes, in 30 interphase cells from each of CHP-212, LA-N-5, and MC-IX, both by serial confocal sections  and a single wide confocal section . The average difference in DM copy number per cell by 2D analysis compared to 3D analysis was very low in each cell line up to a copy-number of approximately 100  were tested against a hypothetical binomial segregation, using the cell with the DM copy-number closest to its own as its presumed post-mitotic sister cell. It has been shown that DMs separate into single minutes during G1-phase One potential explanation of non-binomial DM segregation is detachment of multiple DMs at anaphase, leading to a decreased number of DMs in one of the daughter cells. Further image analysis of the ana- and telophase cells with non-binomial segregation showed that these cells rarely exhibited DMs that detached from the chromosomes, and no more than 4 DMs were observed to be detached in any single cell. This argued against loss of DMs through micronucleus formation as a mechanism for non-binomial segregation. However, the ana- and telophase cells with a non-binomial DM distribution had a higher median DM copy-number than those with a binomial distribution . Based on this, we selected 10 ana-telophases with a DM copy-number >50 from each cell line for a detailed analysis by confocal microscopy. This showed that all of the totally 30 analyzed ana-telophases exhibited at least one cluster of DMs at or adjacent to the chromosome termini . The preMYCN FISH detection in ana-and telophase cells. Analysis of the DMs that remained attached to chromosomes, often arranged in clusters, indeed showed an elevated frequency of segregations deviating (P<0.01) from the binomial distribution compared to unexposed cells, 2% after 0 Gy, 22% after 2 Gy and 24% after 4 Gy  were extremely rare in our model systems, indicating some degree of selection against cells with an extremely high DM copy-number. Based on this, we hypothesized that cells with a higher DM copy-number would divide more frequently up to a certain copy-number level, where the selection pressure would continuously shift to become less favourable  hybrid nuclei. Human- and mouse- specific repetitive DNA sequences and strategy . In thesstrategy . In the strategy . Thus, DMYCN amplification is the strongest biological predictor of outcome in NB patients MYCN copy-numbers can be highly heterogeneous in NB cell populations. Such genetic heterogeneity may reflect different tumour clones and its role is believed to be under-recognized and underestimated in neuroblastoma biology MYCN copies in vivo do not separate at anaphase, but remain as double circles until G1-phase In the present study, we show that DMs in NB cells exhibit a regular topographic fluctuation during the cell cycle, translocating rapidly from the nuclear interior to the periphery of the prometaphase rosette at the initiation of cell division, attaching close to the telomeric repeat sequences. For the vast majority of DMs, this attachment was retained from prometaphase through telophase. After reconstitution of the interphase chromatin structure, the DMs relocated to the nuclear interior, possibly to occupy positions at the periphery of chromosome territories n independent events, each of which yields one out of two possible outcomes with the probability p. In the context of DM segregation, n corresponds to the DM copy-number at metaphase. Because we assumed that each DM was equally likely to segregate to one of the daughter cells as to the other, p was set to 0.5. Our finding that DM copy-numbers can be described by a simple binomial distribution has at least three implications for the evolution of MYCN amplicons in NB cells: (1) MYCN-copies in DMs segregate independently of each other in a random fashion up to a certain copy-number level. (2) When this level has been reached, the random binomial model is no longer applicable; our experimental data indicate that this level is determined by the copy-number at which DMs start to interact with each other to form clusters, presumably because of the spatial restrictions of the mitotic figures. (3) The modal DM copy-number in the tumour cell population reflects the number of MYCN-copies conferring the greatest proliferative advantage under these cells' specific growth conditions. In the established cell lines and tumours used in the present study, this number varied between 25 and 50 DMs.We showed that the skewed frequency distributions of DM copy-number could be modelled by combining binomial mitotic segregation with a selection pressure favouring cells with a higher DM copy-number up to a certain level. The binomial distribution is the discrete probability distribution of the numbers in a sequence of MYCN-copy number after cell division, potentially leading to dysregulation of MYCN-dependent intracellular signalling systems and apoptosis MYCN amplification confers a proliferative advantage We cannot completely exclude that non-binomial segregation of clustered DMs also has a role in generating the skewed frequency distribution found in tumour bulk populations. However, non-binomial segregations were relatively rare in our material. Furthermore, they would be more likely to generate discrete sub-populations at non-modal copy-numbers rather than a continuous, skewed distribution. Our findings do not unequivocally explain the negative selection pressure at higher DM copy-numbers, although it points to clustering as a cause of non-binomial segregation at high copy-numbers. Such mitotic clustering should cause dramatic shifts in per se. A previous study has demonstrated that DMs are typically located at the periphery of folded chromosome territories in interphase nuclei Our findings do not explain the molecular mechanisms underlying the topographical fluctuation of DMs during the cell cycle. The co-localisation of DMs and telomeric TTAGGG-repeats at mitosis could suggest that DMs bind to parts of the telosome multiprotein complex. However, disruption of telomere stability by telomere shortening induced by MST-312 failed to perturb the binomial segregation of DMs at cell division. Even though our studies do not fully exclude that DMs bound to the telomeres that remained intact after telomerase inhibition, our data speak against a specific interaction between DMs and the telosome complex. This was further supported by the finding that not all DMs co-localised with telomere repeats at cell division and also by our confocal microscopy and AFM images showing that several DMs were localised to interstitial segments close to the termini, rather than to the termini MYCN amplicon segregation, we have now acquired a precise standard against which the effects of potential anti-neuroblastoma drugs acting through MYCN amplicon elimination can be compared. The mechanisms by which DMs bind to chromosomes in NB clearly need to be studied further, particularly as the molecules participating in this attachment would be attractive therapeutic targets. If the binding of DMs to chromosomes could be efficiently, and specifically, inhibited, this could be an efficient way of eliminating MYCN amplicons from NB cells, thus causing growth reduction and apoptosis The present study is one of a few showing a specific mechanism causing genetic heterogeneity by random events, which in turn can form the substrate for selection according to Darwinian principles, ultimately leading up to oncogene amplification and an increased proliferation of tumour cells. Furthermore, by clarifying the statistical principles of The study was reviewed and approved by the Ethics Review Board at Lund University Hospital, Sweden. The NB cell-lines CHP-212 and LA-N-5, the neuroepithelioma cell-line MC-IX, and mouse 3T3 cells were obtained from the American Type Culture Collection. Chromosome preparations from NB tumour biopsies and bone marrow infiltrates were obtained from the Department of Clinical Genetics in Lund. Cell culturing, harvest and chromosome preparation for FISH were according to standard methods MYCN and MYC  and telomeric repeat sequences by fluorescein-conjugated (CCCTAA)3 peptide nucleic acid probes as described DMs were detected by single-copy probes for Cells were fixed according to Solovei et al. Combined AFM and optical microscopy investigations were performed using a JPK Nanowizard II AFM with the JPK Life Science stage mounted on an inverted optical microscope . For all measurements, a Nikon CFI Plan Apo VC 60x, 1.40 DIC oil immersion objective was used. For epi-fluorescence a mercury light source was used in combination with standard filter blocks: DAPI , TRITC . A standard CCD camera was used for the digital imaging acquisition. Prior to AFM imaging, the samples were inspected by phase contrast microscopy to locate the regions of interest as well as for precise location of the AFM tip. The cantilever was placed above the selected region using the positioning screws on the stage before approaching the tip to the sample surface. For AFM imaging, standard noncontact cantilevers were used in intermittent contact mode to reduce the lateral forces during scanning. The scan speeds were typically 0.3–0.5 Hz when acquiring images with 512×512 pixels. AFM and FISH images were superimposed using AdobePhotoshop.Cytoplasmic membrane fusion was achieved in sub-confluent cultures containing CHP-212 and 3T3 cells at equal proportions by washing in serum-free medium, followed by treatment with 50% polyethylene glycol  for 2 min. PEG-treated cells and untreated control cultures were then grown in DMEM∶F12 1∶1 supplemented with 10% foetal bovine serum and antibiotics for 3–4 days before harvest.The MST-312 telomerase inhibitor  was diluted in DMSO and used at a noncytotoxic concentration of 1.0 µmol/L (25). To maintain a stable concentration of MST-312, the medium was changed every third day. Cultures exposed only to DMSO were cultured in parallel as controls. Colony formation assay was done by plating 10 000 cells on chamber slides. The rate of population doubling was measured by colony formation assay at regular intervals with colonies defined as groups of >10 cells.The time between divisions for a cell with n DMs was set to 1+10/(1+n)+nˆ2/10000, where the term 1 was the lower limit of the inter-mitotic time, and the term 10/(1+n) simulated the growth advantage of cells with high DM copy-numbers. 1/(n+1) was used instead of 1/n so that cells with 0 DMs would obtain a finite time to the next division. The term nˆ2/10000 was used to simulate a penalty on cells with very high DM counts. After each mitotic cycle, the n for each cell was doubled and then distributed randomly according to a binomial function between the two daughter cells. To keep computations limited we capped the cell population at 100,000 by random deletion. Each simulation was initiated with a single cell starting with a DM count equal to 1 at time zero. Matlab  programming was used to simulate the growth of each population according to these rules.Figure S1Single cell colonies. DM interphase copy-number distributions in single-cell-derived colonies from LA-N-5, CHP-212, and MC-IX. Scored interphase nuclei correspond to red and grey circles, respectively, of which only the former were included in the statistical analysis (see text). Variations in DM copy-number that were not explained (P<0.01) by a binomial distribution are marked by arrows.(0.57 MB TIF)Click here for additional data file.Figure S2DM copy-numbers. The DM frequency distribution in near-diploid metaphase cells in the CHP-212 and MC-IX cell lines, and in biopsies from one primary NB (Patient 1), and one bone marrow NB metastasis (Patient 2). Similar to SK-N-5, the distributions are skewed towards higher copy-numbers and differ from a normal distribution .(0.10 MB TIF)Click here for additional data file.


Although the most frequent extra-pulmonary manifestations of respiratory syncytial virus (RSV) infection involve the cardiovascular system, no data regarding heart function in infants with bronchiolitis associated with RSV infection have yet been systematically collected. The aim of this study was to verify the real frequency of heart involvement in patients with bronchiolitis associated with RSV infection, and whether infants with mild or moderate disease also risk heart malfunction.A total of 69 otherwise healthy infants aged 1-12 months with bronchiolitis hospitalised in standard wards were enrolled. Pernasal flocked swabs were performed to collect specimens for the detection of RSV by real-time polymerase chain reaction, and a blood sample was drawn to assess troponin I concentrations. On the day of admission, all of the infants underwent 24-hour Holter ECG monitoring and a complete heart evaluation with echocardiography. Patients were re-evaluated by investigators blinded to the etiological and cardiac findings four weeks after enrolment.Regardless of their clinical presentation, sinoatrial blocks were identified in 26/34 RSV-positive patients (76.5%) and 1/35 RSV-negative patients (2.9%) (p < 0.0001). The blocks recurred more than three times over 24 hours in 25/26 RSV-positive patients (96.2%) and none of the RSV-negative infants. Mean and maximum heart rates were significantly higher in the RSV-positive infants (p < 0.05), as was low-frequency power and the low and high-frequency power ratio (p < 0.05). The blocks were significantly more frequent in the children with an RSV load of ≥100,000 copies/mL than in those with a lower viral load (p < 0.0001). Holter ECG after 28 ± 3 days showed the complete regression of the heart abnormalities.RSV seems associated with sinoatrial blocks and transient rhythm alterations even when the related respiratory problems are mild or moderate. Further studies are needed to clarify the mechanisms of these rhythm problems and whether they remain asymptomatic and transient even in presence of severe respiratory involvement or chronic underlying disease. The most frequent extra-pulmonary manifestations of respiratory syncytial virus (RSV) infection involve the cardiovascular system , and incAs clinically relevant heart problems are usually found in infants whose bronchiolitis is severe enough to require mechanical ventilation ,6, it isThe aim of this study was to verify the real frequency of heart involvement in patients with bronchiolitis associated with RSV infection, and whether infants with mild or moderate disease also risk heart malfunction.This prospective study was carried out at the Department of Maternal and Pediatric Sciences of the University of Milan, Italy, during the winter seasons 2007-2008 and 2008-2009. The protocol was approved by the local Ethics Committee, and written informed consent to study participation was obtained from the patients' parents or legal guardians.The study involved otherwise healthy infants aged 1-12 months who were admitted to hospital because of bronchiolitis during the study period. The exclusion criteria were the presence of a chronic disease increasing the risk of complications of respiratory infection, including chronic disorders of the pulmonary or cardiovascular system, chronic metabolic disease, neoplasms, kidney or liver dysfunction, hemoglobinopathies, immunosuppression, and genetic or neurological disorders. There was no refusal to participate.Upon admission, the infants' demographic characteristics and medical history were systematically recorded using standardised written questionnaires and, after a complete physical examination, the subjects with a diagnosis of bronchiolitis based on well-established criteria  were enrUpon enrolment, Virocult  nasopharyngeal swabs were used to collect specimens for the detection of RSV, and a blood sample was drawn to assess troponin I concentrations. On the basis of our previous experience in children with bronchiolitis in which we showed that RSV was the main cause of acute episodes in hospitalized children , in thisDuring their hospital stay, the infants' clinical signs and symptoms were monitored daily. They were treated with oxygen when saturation was ≤ 95%, and received inhalatory bronchodilators, steroids, antibiotics, intravenous fluids and chest physiotherapy on the basis of the judgement of the attending pediatrician. They were discharged when they were able to maintain > 95% oxymetry without oxygen, but their parents were asked to bring them immediately to the study centre if there were any recurrent or worsening signs and symptoms.The medical history, general physical condition and clinical symptoms of each patient were re-evaluated by investigators blinded to the etiological and cardiac findings four weeks after enrolment. During this follow-up visit, the patients' history of respiratory tract infections was carefully assessed and 24-hour Holter ECG monitoring 24 hours was repeated.The Virocult nasopharyngeal swabs were tested by means of previously described real-time polymerase chain reaction (PCR) for RSV types A and B -24, withRNA was amplified in a single tube, two-step reaction using Taqman reverse transcriptase and PCR core reagent kits  and an ABI 7700 or ABI 7500 sequence detection system (Applied Biosystems). A cultured positive control virus was used for each assay. On the basis of proficiency testing data, the sensitivity of each assay was estimated to be less than 500 copies ⁄ mL.To evaluate myocardial damage, serum troponin I levels were measured using the Abbott AxSYM system  at the time of hospital admission, and were considered indicative of myocardial damage when they were > 1.2 μg/L. The measurements had a coefficient of variation of 10%, and the lower detection limit was 0.3 μg/L.Three-channel Holter monitors  were positioned immediately after hospital admission, and 24-hour recordings were obtained. After the skin had been prepared, the electrodes were placed to record leads II, V1 and V5; a 1 mV calibration signal was also recorded. The built-in clock started after the electrodes had been attached.A commercial Holter analysis software  was used to analyse rhythm and heart rate variability  from the Holter tapes. QRS was detected using a level detector, but was manually over-read by a physician. All of the tapes were edited in order to assure the accuracy of the QRS classification. Ectopic beats, noisy data, and artifacts were manually identified and excluded from the HRV analysis. Non-stationarities were avoided by means of trigger adjustment. Average hourly heart rates were determined from the computerised Holter scanner, and maximum, minimum, and mean 24-hour heart rates  were calculated for each subject.The time-domain parameters measured from the Holter tapes were: 1) the average of all normal-to-normal beats  (mean heart rate); 2) the SD of all NN intervals (SDNN); 3) the SD of the average of NN intervals in all 5-minute segments of the 24-hour recording (SDANN); 4) the mean of the standard deviation in all 5-minute segments of the 24-hour recording (ASDNN); 5) the square root of the mean of the squares of the differences between adjacent NN intervals (rMSSD); and 6) the percentage of > 50 msec differences between adjacent NN intervals. Frequency-domain heart rate variability was also determined, including low-frequency power , high-frequency power , and the LF/HF ratio .The echocardiographic studies were made using a real-time ultrasound imaging system system (Acuson Sequoia 512) equipped with 3-, 5-, 7 and 10A, MHz transducers. The echocardiographic measurements were made using standard techniques .M-mode measurements were made in accordance with the recommendations of the Cornmittee of M-Mode Standardization of the American Society of Echocardiography , and werThe flow velocities across the mitral, tricuspid, aortic and pulmonary valves were recorded from standard pericordial and subcostal positions using pulsed-wave and continuous-wave Doppler transducers.vs RSV-negative), the continuous data were analysed using a two-sided Student's test if they were normally distributed (on the basis of the Shapiro-Wilk statistic) or a two-sided Wilcoxon rank-sum test if they were not. For the comparison within group , the continuous data were analysed using a paired two-sided Student's test or signed-rank test, as appropriate. Categorical data were analysed using contingency table analysis and the chi-square or Fisher's exact test, as appropriate.Continuous variables are given as mean values ± SD, and categorical variables as numbers and percentages. For the comparison between groups  RSV-positive and 35 (50.7%) RSV-negative. Table Table vs RSV-negative patients). Mean and maximum heart rate were significantly higher in the RSV-positive infants (p < 0.05). Among the HRV time-domain parameters, the prevalence of LF periods was significantly higher in the RSV-positive infants (p < 0.05) as was the LF/HF ratio (p < 0.05). Twenty-four hour Holter ECG monitoring 28 ± 3 days later demonstrated the complete regression of the heart abnormalities in all of the RSV-positive infants: no block was recorded and their HRV parameters were similar to those recorded in the RSV-negative patients during the acute phase of the disease and during the convalescent period.Table Table The results of this study indicate that bronchiolitis during the course of RSV infection is frequently associated with sinoatrial blocks, an increase in absolute heart rate, and an increase in the LF component of HRV. All of these findings seem to be specific of RSV infection because they have not been demonstrated in children with bronchiolitis caused by a different infectious agent. Our data confirm and extend what has been previously reported by other authors who have found that RSV infection can be associated with cardiac rhythm alterations ,9,28.Sinoatrial blocks are rare in pediatrics but, when symptomatic, have been described in otherwise healthy children and patients with heart malformations or myocarditis . To the One limitation of this study is that the population is too small to allow any definite conclusions to be drawn and so further studies of larger series are needed. It seems to be particularly important to study more severe cases in order to verify whether more significant lung involvement can precipitate arrhythmias and cause more serious clinical problems. It is interesting that in our population all the three cases of severe bronchiolitis showed a sinoatrial block. The very low number of subjects with severe infection could have limited the statistical power to detect between group differences according to disease severity. Another limitation is the fact that only RSV has been searched in respiratory secretions. Despite it represents the absolute main cause of bronchiolitis in infants and in various studies it has been detected as single pathogen in more than 60% of the cases ,22, it cOur data support the hypothesis that the heart involvement diagnosed in some cases of bronchiolitis associated with RSV infection -8 could RSV seems associated with sinoatrial blocks and rhythm alterations even when the resulting respiratory difficulties are mild or moderate. Further studies are needed to clarify the mechanisms of these rhythm problems and whether they remain asymptomatic and transient even in presence of severe respiratory involvement or chronic underlying disease. Finally, as RSV can cause respiratory illnesses other than bronchiolitis, further researches specifically aimed at defining the relationships between RSV and the heart are urgently needed regardless of the clinical picture.: Average of all normal-to-normal beats; (bpm): beats per minute; (cTnI): cardiac troponin I; (CPK): creatine phosphokinase; (HRV): heart rate variability; (HF): high-frequency power; (LDH): lactate dehydrogenase; (LVID): left ventricular internal dimensions in diastole; (LVIS): left ventricular internal dimensions in systole; (LF): low-frequency power; (ASDNN): mean of the standard deviation in all 5-minute segments of the 24-h recording; (nu): normalised units; (PICUs): pediatric infectious disease units; (pNN50): percentage of differences between adjacent NN intervals of > 50 msec; (PDV): phocine distemper virus; (PCR): polymerase chain reaction; (RSV): respiratory syncytial virus; (RVID): right ventricular internal dimension in diastole; (SGOT): serum glutamyl oxaloacetic transaminase; (SGPT): serum glutamic pyruvic transaminase; (rMSSD): square root of the mean of the squares of the differences between adjacent NN intervals; (SD): standard deviation; (SDNN): standard deviation of all NN intervals; (SDANN): standard deviation of the average of NN intervals in all 5-minute segments of the 24-h recording.The authors declare that they have no competing interests.SE and NP designed the study and co-wrote the manuscript.PS and SG performed the cardiologic studies.PB assisted in the interpretation of cardiologic data.SB carried out the real-time PCR.CT and LG visited the patients during hospitalization.ET performed the follow-up visits.CG performed the statistical analysis.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/305/prepub


As a simple example, a recessive locus that results in fetal lethality will result in live-born individuals sharing more alleles at this locus than expected under Mendel's laws. This could result in apparent linkage of the phenotype of 'being alive' to such a chromosomal regions. Further, this could result in false-positive linkage when 'affected-only' parametric or non-parametric linkage analysis is performed. Similarly, loci demonstrating TRD may be detectable in family-based association tests as deviant transmission of alleles. Therefore, TRD could result in confounding of family-based association studies of diseases. The Framingham Heart Study data available for Genetic Analysis Workshop 16 is a suitable dataset to determine whether there are loci in the genome that reveal TRD because of the large number of individuals from families, the high-resolution genotyping, and the population-based nature of the study. We have used both genome-wide linkage and family-based association methods to determine whether there are loci that demonstrate TRD in the Framingham Heart Study. Family-based association analysis identified thousands of loci with apparent TRD. However, the vast majority of these are likely the result of genotyping errors with application of strict quality control criteria to the genotype data, and automated inspection of the intensity plots, we identify a small number of loci that may show true TRD, including rs1000548 in intron 6 of S-antigen  on chromosome 2 ( A critical assumption for the majority of genetic mapping approaches (including both linkage and family-based association) is that Mendel's law of segregation is obeyed. Transmission-ratio distortion (TRD) refers to the deviation from the expected Mendelian inheritance of alleles. Violation of this assumption could result in false-positive linkage, particularly within 'affected-only' or 'non-parametric' linkage analysis frameworks. Futhermore, within a family-based association design, the presence of TRD could produce spurious association if transmissions are only assessed to affected, but not unaffected offspring. In addition, it is feasible that the presence of TRD could also reduce the power to detect true disease loci. The presence of TRD in humans has been addressed in only a few studies, using either linkage  or familFor a variety of reasons, including that of statistical power, the majority of genome-wide association studies have used a case-control design, which is not able to detect loci that are subject to TRD. However, some studies are employing a family-based design, but it is typical for them to study only affected offspring, and they are thus susceptible to identifying loci that demonstrate TRD and falsely concluding that they are associated with the disease of interest. Unless unaffected sibs are genotyped, one cannot determine whether association signals are the result of confounding by TRD. Therefore, we took advantage of the large sample size, pedigree-based design and genome-wide genotyping of the Framingham Heart Study Problem 2 data from Genetic Analysis Workshop 16 (GAW16) to determine whether we could identify loci demonstrating TRD.We used data from Affymetrix 500 k and 50 k single-nucleotide polymorphism (SNP) datasets from Problem 2 of GAW16, the Framingham Heart Study. Genotype data were called by the data providers using BRLMM , but no p-value > 0.05, individual genotype missing rate <5%, SNP missing rate <2%, pairwise r2 < 0.05, and Mendelian error rate <5%. Individuals from Cohort 1 were not used in the analysis, therefore large pedigrees were split into smaller pedigrees using the R kinship package ) to determine whether there are regions in the genome linked to the phenotype of 'being alive in the last generation' (Merlin v 1.1.2) . We dealfunction ) to allop > 10-5.We also performed family-based association tests  to examine the transmissions of alleles for all SNPs across the genome to all genotyped individuals in the dataset using PLINK v1.02 ,8 with tGenome-wide linkage analyses used ~5 k SNPs from 1,028 pedigrees that were informative. There were no loci that met genome-wide criteria for significant linkage.p < 10-8, which was an unexpectedly large number. However, when we investigated this further, we suspected that the majority of these results were false positives due to genotyping error. It has been reported previously that, in the presence of certain common types of genotyping error, there is a bias to excess transmission of the major as opposed to minor allele for SNPs [p < 10-8, HWE p > 10-5, and MAF>1% in which the major allele showed excess transmission. This compared to only 21 SNPs using the same criteria in which the minor allele showed excess transmission.Genome-wide TDT analysis was performed and identified 2,722 autosomal SNPs with TDT for SNPs . Indeed,p < 0.0001).To confirm our suspicions that genotyping error was the major cause of the large number of positive results, we took advantage of the fact that it is more difficult to detect Mendelian errors for SNPs with lower MAF . This wop < 10-5), and for these 4,501 SNPs we ran automated cluster plot analysis (ACPA) [p-value>10-4. Using a criteria for genome-wide significance of p < 10-8, only one SNP was predicted by the ACPA procedure to have good quality genotype clustering, rs1000548  are provided in Table p > 0.08).Visual inspection of the cluster-plots of thousands of SNPs is labor intensive, so we next investigated whether we could use automated methods to help distinguish which SNPs had good quality genotype calls. We then applied a less stringent criteria for TRD  . We limiThe results of TDT analyses performed here have highlighted the problems of using high-throughput genotype data with even a small proportion of genotyping errors to detect phenomenon such as TRD. The gross over-transmission of the common allele for SNPs with a pattern consistent with TRD, and the marked allele frequency difference between them and the SNPs where the minor allele shows excess transmission, are consistent with genotyping error being the major force behind the unexpectedly large number of apparent positive results. Further contributing to the bias described by Mitchell et al.  in whichIn addition to the complexities that have arisen in the interpretation of our analysis, there is concern that the use of HWE as a criterion to filter SNPs for the analysis of TRD is a double-edged sword. Some SNPs showing true TRD may also deviate significantly from HWE because of violation of the selection assumption, and may end up being removed from datasets in an attempt to remove genotyping errors. Similarly, automatic exclusion of SNPs with low MAF may bias against the detection of true TRD loci because it is likely that because of negative selection, SNPs which show TRD tend to have low MAF. Another caveat of this study is that at each of the eight regions with evidence for TRD Table , there iCTDP1  on chromosome 18; autosomal recessive mutations in this gene have been shown to results in 'congenital cataracts facial dysmorphism neuropathy' (CCFDN), a developmental disorder prevalent in Roma Gypsies [SAG  have been found in Oguchi disease, a rare autosomal recessive form of night blindness [SAG. It may be that in populations similar to Framingham, variation in these genes contributes to phenotypes that can result in TRD, including the failure of fertilization, implantation, or the differential survival of fetuses. Identifying loci that demonstration TRD could provide insight into the mechanisms of the processes.There are some interesting genes near the SNPs in Table  Gypsies . Similarlindness . In thisACPA: Automated cluster plot analysis; GAW: Genetic Analysis Workshop; HWE: Hardy-Weinberg equilibrium; MAF: Minor allele frequency; NPL: Non-parametric linkage; TDT: Transmission-disequilibrium test; TRD: Transmission-ratio distortion; SNP: Single-nucleotide polymorphism;The authors declare that they have no competing interests.ADP, CI-R and SBB conceived of the idea. DW, DP, and YJY performed the linkage and association analysis. AS ran the ACPA analysis. ADP wrote a draft of the manuscript which all authors edited, read and approved the final manuscript.


Experimental and epidemiological evidence suggests that homocysteine (tHcy) may be a causal risk factor for atherosclerosis. B-vitamin supplements reduce tHcy and improve endothelial function in short term trials, but the long-term effects of the treatment on vascular structure and function are unknown.6 25 mg and vitamin B12 0.5 mg) in the prevention of vascular events in patients with a history of stroke. We measured carotid intima-medial thickness (CIMT) and flow-mediated dilation (FMD) at least two years after randomisation in 162 VITATOPS participants. We also conducted a systematic review and meta-analysis of studies designed to test the effect of B-vitamin treatment on CIMT and FMD.We conducted a sub-study of VITATOPS, a randomised, double-blind, placebo-controlled intervention trial designed to test the efficacy of long term B-vitamin supplementation . Post-treatment CIMT  and FMD  did not differ significantly between groups. A meta-analysis of published randomised data, including those from the current study, suggested that B-vitamin supplements should reduce CIMT  and increase FMD . However, the improvement in endothelial function associated with homocysteine-lowering treatment was significant in short-term studies but not in longer trials.Although short-term treatment with B-vitamins is associated with increased FMD, long-term homocysteine-lowering did not significantly improve FMD or CIMT in people with a history of stroke.Clinical Trial Registration URL: Trial Registration number: 12605000005651 An elevated plasma homocysteine concentration (tHcy) is associated an increased risk of myocardial infarction and stroke -4. It re6 and vitamin B12 would reduce CIMT and increase FMD in subjects with a history of stroke. We also carried out a systematic review and meta-analysis to obtain an estimate of the effect of B-vitamin treatment on these vascular measurements based on the totality of published randomised evidence.The aim of this study was to determine whether long-term homocysteine-lowering treatment with folic acid, vitamin BThe study was conducted in accordance with the Declaration of Helsinki. The Royal Perth Hospital Ethics Committee approved the study protocol and each subject gave written informed consent prior to taking part.Study subjects were recruited from participants in the Vitamins TO Prevent Stroke (VITATOPS) trial. VITATOPS is a large, international, randomised, double-blind, placebo-controlled clinical trial designed primarily to examine the efficacy and safety of B-group vitamins in the prevention of stroke, myocardial infarction or death from any vascular cause among patients with previous stroke or transient ischemic attack (TIA). The inclusion and exclusion criteria for the VITATOPS trial have been reported previously . VITATOPThe current study was a cross-sectional single-centre sub-study of the VITATOPS trial. All VITATOPS participants enrolled in Perth, Australia between 1998 and 2003 were eligible for inclusion (n = 532). Subjects who had died, withdrawn from VITATOPS, were severely handicapped or had moved away from Perth were excluded (n = 189). Recruitment letters were sent to all subjects suitable for inclusion (n = 343). Subjects who responded positively to the letter were contacted by telephone and enrolled in this study (n = 173). The ethics approval for the project stipulated that we should not contact subjects who did not respond to the invitation letter, so non-responders were not followed up.6 25 mg, and vitamin B12 500 μg or a matching placebo. A central 24-hour telephone service and an interactive website used random permuted blocks stratified by hospital to allocate a treatment pack number.Participants in the VITATOPS trial were randomly assigned at baseline to a single daily tablet containing folic acid 2 mg, vitamin BWe aimed to determine whether long-term (greater than two years) homocysteine-lowering treatment with B-vitamin supplements would reduce CIMT and increase FMD compared with placebo in subjects with a history of stroke or TIA.6 and B12, total cholesterol, high- and low-density lipoprotein (HDL and LDL) cholesterol, triglycerides, creatinine and glucose.CIMT and FMD were the outcome measures chosen for this sub-study. The vascular outcomes were not measured at baseline and were assessed at a single time point at least two years after randomisation. All participants attended a morning appointment after an overnight fast. They were also asked to withhold their morning medications, including the VITATOPS tablet, and to refrain from smoking and drinking alcohol or caffeine-containing beverages for at least six hours prior to the study appointment. Age, gender, details of the primary event, medications, cardiovascular risk factors and smoking history were recorded and blood pressure, height, weight, waist and hip girth and FMD and CIMT were measured. A fasting blood sample was collected to assess tHcy, red blood cell folate, serum BCIMT and FMD were measured using techniques described previously ,15. BrieFMD was measured in the left arm except in subjects with a left hemiplegia (n = 18). B-mode images of the brachial artery were obtained and recorded continuously for one minute to assess baseline arterial diameter. A rapid inflation/deflation pneumatic cuff placed around the forearm was inflated to 250 mmHg for five minutes. Post cuff-deflation images were recorded continuously for two minutes to capture peak arterial dilation. When fifteen minutes had elapsed, a second scan was recorded to re-establish baseline diameter. 400 μg of sub-lingual glyeryl trinitrite (GTN) was administered to the subject and images were recorded for eight minutes. FMD and GTN-mediated dilation were measured off-line using edge-detection software designed to minimise observer biases. A single observer blinded to treatment status performed all measurements. The mean intra-observer CV's for FMD and GTN-mediated dilation measured by this technique are 6.7% and 3.9% respectively .We aimed to recruit sufficient subjects from the Perth VITATOPS cohort to provide 80% power (α = 0.05) to detect differences of 0.1 mm in mean CIMT and 2% in FMD between the vitamin and placebo groups. Post-hoc calculations for a one-sided test showed that the study had 80% power (α = 0.05) to detect a reduction in mean CIMT of 0.07 mm and an absolute increase in FMD of 1.6%.As the FMD data had a non-normal distribution and multiple zero values, we used a two-sample rank sum test (Mann-Whitney) to compare the median FMD in the treatment and placebo groups. Two-sample t-tests were used to compare all normally distributed variables and results are reported as mean ± standard deviation. Variables with log-normal distributions were log-transformed and results for these variables are reported as geometric mean . Proportions were compared using a normal approximation to the binomial. Effect size was calculated as the standardised difference between treatment group means (Cohen's d) using Hedges' correction for bias. Minitab  and SPSS  were used for statistical analyses. A single observer blinded to treatment group conducted the statistical analysis.carotid intima media or intima medial thickness or IMT) AND (folate or folic acid or folic) for CIMT papers and  AND (folate or folic acid or folic) for the FMD papers. Figure We conducted a systematic review and meta-analysis of randomised controlled trials reporting the effect of homocysteine-lowering treatment with B-vitamin supplements on FMD and CIMT. Papers were identified in the Medline (1950 to January 2008) and EMBASE (1988 to January 2008) databases and with a manual search of citations in the relevant retrieved papers. The search strategy was  and two from the vitamin group (food intake and cigarette smoking less than six hours prior to the study session).6 and serum B12 levels at follow-up than the placebo group.The demographic and clinical characteristics of each group at baseline and follow-up are presented in Table The vascular outcome measurements are presented in Table 2 = 84.1%) in vitamin-treated subjects compared with placebo . Using a fixed-effect versus a random-effects model did not substantially alter the pooled estimate. However, removing the Vianna (2007) data from the analysis reduced the pooled estimate to non-significance  [We identified six randomised placebo-controlled studies that measured the effect of B-vitamin treatment on CIMT. The pooled estimate from these studies was a significant reduction in CIMT of 0.13 mm  in vitamin-treated subjects compared with placebo  and FMD  in our study overlap with confidence intervals from the meta-analyses of -0.25 mm to -0.01 mm and 0.73% to 2.23% (current data excluded), indicating that our results are consistent with pooled data from similar randomised studies. In a recent systematic review and meta-analysis, Lorenz et al. report that a difference of 0.1 mm in CIMT predicts a 10% to 15% reduction in the risk of future MI and a 13% to 18% reduction in the risk of future stroke . To the 2 = 85%), are strongly influenced by a single study and are likely to have been influenced by publication biases. A large study (n = 819) that has not yet published detailed data has recently reported finding no difference between placebo and treatment groups in mean CIMT after three years of treatment with folic acid [One explanation is that the results of the CIMT meta-analysis are unreliable. Only a small number of studies have measured the effect of B-vitamin treatment on CIMT , the results show considerable heterogeneity  . A possiIt is possible that homocysteine-lowering treatment may prove more effective in individuals without established vascular disease, as a recent meta-analysis of clinical trial data suggests that the incidence of new ischemic stroke is significantly reduced by B-vitamin supplementation whilst recurrent stroke is not . HoweverOur sub-group analysis also indicates that the significant improvements in FMD associated with short-term folic acid treatment are not sustained in longer-term studies Figure . WhetherA more controversial explanation for the discrepancy between the improvements in FMD and CIMT in our meta-analysis and the negative results from the clinical trials is that FMD and CIMT are not particularly reliable or sensitive indicators of cardiovascular risk. Although FMD and CIMT are widely and enthusiastically accepted as surrogates for vascular events, the evidence linking these measurements with hard clinical endpoints is relatively weak and the small changes in FMD and CIMT associated with B-vitamin treatment in our meta-analyses may actually have limited independent prognostic significance ,31.Our study has several strengths, namely its large size, long duration and randomised, placebo-controlled, double-blind design. Ours is the first study to investigate the effects of B-vitamin supplementation on FMD and CIMT in people with a history of stroke and TIA. By conducting a systematic review and meta-analysis, we were able to estimate the effect of B-vitamin treatment on FMD and CIMT based on the totality of available randomised evidence. This additional analysis puts our results in proper context and allows for the addition of more randomised data as they are published.Our study also has limitations. We did not measure the FMD or CIMT prior to randomisation and were thus unable to adjust the post-treatment change in these measurements for baseline values. The randomisation process should have ensured that both groups had similar CIMT and FMD at baseline, making it statistically valid to estimate the effect of treatment by testing only the follow-up measurements. By chance, however, the group randomised to B-vitamins had a lower mean tHcy at baseline than the group randomised to placebo. Although the two groups were similar in all other clinical characteristics, we cannot be sure that the mean CIMT and FMD were actually the same in both groups prior to treatment. However, as tHcy is positively associated with CIMT and negatively with FMD, any baseline differences in these measurements between the groups should have increased rather than reduced the probability of finding improved FMD and CIMT in the vitamin group at follow-up.A further limitation of the study is that, due to ethical constraints, we did not contact subjects who failed to respond to the invitation letter. We collected usable data from only 30% of the subjects originally randomised, approximately half of the subjects identified retrospectively as eligible for inclusion. Apart from a higher proportion of males and fewer haemorrhagic strokes, participants did not differ significantly from the eligible non-participants in demographic or clinical characteristics and thus appeared to be broadly representative of the larger cohort Table . It is lSelecting subjects who had survived for at least two years after randomisation without severe disability may also have influenced our results. If subjects with a low FMD or high CIMT randomised to the vitamin group had improved disability-free survival compared with similar subjects randomised to placebo, they may have been over-represented in the vitamin-group at follow-up, reducing our chances of finding a between-group difference in the vascular outcomes. However, we found no difference in the number of subjects in each group excluded from the current study due to death or disability, making it unlikely that this potential source of bias substantially altered our results.It is not clear that our negative result can be extrapolated to people with a history of stroke in the general population. The majority of our subjects were on optimal medical treatment, which may have made it difficult to detect any additional benefit from lowering tHcy. In addition, the subjects were a self-selected group of trial participants and were thus a highly motivated and compliant group of individuals unlikely to be representative of the community at large.Our results indicate that long-term homocysteine-lowering treatment with B-vitamins does not significantly reduce CIMT or increase FMD in people with a history of stroke. In addition, the modest increase in FMD associated with short-term B-vitamin treatment does not appear to be translated into improved vascular structure or sustained in the longer term.CIMT: indicates carotid intima-media thickness; FMD: flow-mediated dilation; GTN: glyeryl trinitrite; tHcy: total plasma homocysteine; VITATOPS: VITAmins TO Prevent StrokeGJH, JE and KJ are all investigators on the VITATOPS trial. The authors declare that they have no competing interests.GJH, JE and KJ are investigators on the VITATOPS trial. LFA, GJH, DJG and KP contributed to the design of the VITATOPS sub-study reported in this paper. KP recruited the study participants, measured FMD and CIT, collected, recorded and analysed the study data and wrote the draft manuscript. GJH, JE, DJG, KJ and LFA all critically reviewed the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Second-generation antipsychotic drugs have been reported to cause fewer incidences of extrapyramidal side effects (EPSs) than typical antipsychotic drugs, but adverse events such as akathisia have been observed even with atypical antipsychotic drugs. Although understanding of the pathophysiology of akathisia remains limited, it seems that a complex interplay of several neurotransmitter systems might play a role in its pathophysiology. The endoplasmic reticulum protein sigma-1 receptors are shown to regulate a number of neurotransmitter systems in the brain.We report on two cases in which monotherapy of the selective serotonin reuptake inhibitor and sigma-1 receptor agonist fluvoxamine was effective in ameliorating the akathisia of patients with schizophrenia treated with the antipsychotic drug aripiprazole.The global score on the Barnes Akathisia Scale in the two patients with schizophrenia treated with aripiprazole decreased after fluvoxamine monotherapy.Doctors may wish to consider fluvoxamine as an alternative approach in treating akathisia associated with antipsychotic drugs such as aripiprazole. Second-generation antipsychotic drugs have been reported to cause fewer incidences of extrapyramidal side effects (EPSs) than typical antipsychotic drugs, but adverse events such as akathisia have been observed even with atypical antipsychotic drugs. Akathisia is one of the most common and distressing EPSs of antipsychotic drugs ,2. The d2+ signalling and cell survival, and have been shown to regulate a number of neurotransmitter systems in the central nervous system [2+ signalling and bioenergetics within the cell [11C]SA4503 and positron emission tomography demonstrated that fluvoxamine binds to sigma-1 receptors in living human brain at therapeutic doses, suggesting that sigma-1 receptors might play a role in the mechanism of action of fluvoxamine [The endoplasmic reticulum protein sigma-1 receptors play a key role in Cas system -8. A recs system ,10. Furtthe cell ,12. A stvoxamine .2 receptors and serotonin 5-hydroxytryptamine (5-HT)1A receptors, and an antagonist at 5-HT2A receptors. The Schizophrenia Trial of Aripiprazole (STAR) study demonstrated a relatively higher incidence of akathisia with aripiprazole compared with placebo or other antipsychotic drugs [Given the important role of sigma-1 receptors in the regulation of neurotransmitter systems, we hypothesised that fluvoxamine may be effective in the treatment of akathisia associated with antipsychotic treatment. Aripiprazole is an antipsychotic drug that acts as a partial agonist at dopamine Dperidone). Here, wThe patient was a 24-year-old woman who met the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) criteria for schizophrenia. Treatment with aripiprazol (12 mg) was initiated; 2 days later, the patient complained of leg restlessness. Her global score on the Barnes Akathisia Scale  was 3 . He showed signs of akathisia after the dose of aripiprazole was increased to 12 mg. His global score on the Barnes Akathisia Scale was 3. Administration of fluvoxamine  rapidly improved the akathisia. He showed no signs of akathisia after the dose of aripiprazole was increased to 24 mg, his body weight decreased, and his mental status was stable.To our knowledge, this is the first report demonstrating that fluvoxamine is effective in the treatment of aripiprazole-induced akathisia of patients with schizophrenia. Furthermore, we have experienced that fluvoxamine is also effective in the treatment of other antipsychotic-induced akathisia in patients with schizophrenia (data not shown). Nonetheless, a randomised double-blind, placebo-controlled study of fluvoxamine will be needed to confirm its efficacy for the treatment of this syndrome. From these case studies, it is unclear whether sigma-1 receptor agonism appears to be irrelevant to the anti-akathitic action of fluvoxamine. In order to confirm the role of sigma-1 receptors in the treatment of akathisia, a randomised double-blind, placebo-controlled study of the selective sigma-1 receptor agonist ) in patients with antipsychotic-induced akathisia would be also of interest.2 receptor blockade [Akathisia is a neurological side effect of antipsychotic medications, which are used to treat various psychiatric disorders such as schizophrenia and bipolar disorders ,2,4. It blockade  althoughblockade ,2. At prblockade -8, it isThese two cases suggest that fluvoxamine may serve as an alternative option in the treatment of antipsychotic-induced akathisia in patients with schizophrenia. More detailed randomised, double-blind studies of fluvoxamine using larger samples should be performed to clarify the role of sigma-1 receptors in the efficacy of fluvoxamine for akathisia.The authors declare that they have no competing interests.TF contributed to the clinical and rating evaluations during the follow-up periods. KH conceived of the study and participated in its study and coordination. All authors read and approved the final manuscript.