Despite mounting evidence of their considerable importance in development, the biochemical properties and nature of MAB-21 proteins have remained strikingly elusive. In addition, genetic studies conducted in C. elegans have established that in double mutants mab-21 is epistatic to genes encoding various members of a Transforming Growth Factor beta (TGF-beta) signaling pathway involved in the formation of male-specific sensory organs.Through Mab21l2 with a TGF-beta signaling pathway in early vertebrate development. We show that the vertebrate mab-21 homolog Mab21l2 antagonizes the effects of Bone Morphogenetic Protein 4 (BMP4) overexpression in vivo, rescuing the dorsal axis and restoring wild-type distribution of Chordin and Xvent2 transcripts in Xenopus gastrulae. We show that MAB21L2 immunoprecipitates in vivo with the BMP4 effector SMAD1, whilst in vitro it binds SMAD1 and the SMAD1-SMAD4 complex. Finally, when targeted to an heterologous promoter, MAB21L2 acts as a transcriptional repressor.Through a gain-of-function approach, we analyze the interaction of Our results provide the first biochemical and cellular foundation for future functional studies of mab-21 genes in normal neural development and its pathological disturbances. In that context, early studies [mab-21 allele as a dominant enhancer of egl-5, a loss-of-function mutation affecting the nematode homolog of the Drosophila homeotic gene AbdominalB. Subsequent studies [mab-21(+) plays a key role in the formation of the nematode male tail. Homozygous mab-21 mutations act cell-autonomously on the identity of a sensory ray, leading to alterations in anteroposterior identities akin to a homeotic transformation. Furthermore, they act non-cell-autonomously on the binary switch between hypodermal and neuroblast specification. Finally, hypomorphic mab-21 mutants exhibit pleiotropic changes affecting movement, body shape and fecundity, suggesting that mab-21(+) also plays key developmental roles outside the male tail region.The mab-21 gene encodes a 41 kD basic protein with no significant homologies to any other published proteins. Homologs of the mab-21 gene have been identified in many other species, from Drosophila to humans. The first vertebrate counterparts of mab-21 were isolated in amniote genomes: a human homolog, MAB21L1, was cloned during a systematic search for transcripts carrying trinucleotide repeats [Mab21l1 within a chromosomal deletion discovered in a patient with autism and language deficit secondary to an auditory processing abnormality [The  repeats  and potemab-21 genes  [MAB21L2 gene, and showed that they encode nuclear proteins [Mab21l1 and Mab21l2 are expressed in largely overlapping territories [Xenopus laevis (Xmab21l2) [Danio rerio [Zmab21l2). Mab21l2 is an early marker of the tectum as well as primitive eye field, optic cup and retina in anamniotes, in agreement with observations previously made in mouse embryos.Our group, in parallel with others, identified two murine Mab21l2) -8 and thproteins  that areritories -7, suggemab21l2) , , while they do not affect body size in double mutants [mab-21 gene transcription by the sma signaling pathway.Some of these important advances point to a critical role for the mab-21 gene family as a whole in early embryogenesis on one hand, and in various aspects of neural development on the other. However, our knowledge on the molecular mechanisms through which mab-21 genes operate remains inadequate at best. Interestingly, by genetic analysis of loss-of-function mutations in nd CET-1 , also knnd CET-1 , and reg mutants . Reportemab-21 – TGF-beta interaction, but stop short of addressing several key questions. What is the molecular counterpart of the genetic interaction described in C. elegans between mab-21 and the sma pathway? Is this interaction conserved in vertebrate development, and what TGF-beta superfamily molecules does it involve? Does it play a relevant role in the establishment of vertebrate dorsoventral (DV) polarity? If so, at what level do MAB-21 and TGF-beta pathways converge into a single regulatory cascade? In the present study, through a gain-of-function strategy, we analyze the molecular interplay between MAB21L2, a vertebrate homolog of C. elegans MAB-21, and TGF-beta superfamily signaling molecules. In particular, we focus on the signaling pathway initiated by BMP4, a ligand structurally related to CET-1/DBL-1, that regulates DV axis formation and numerous key aspects of neural development in chordata.The above genetic studies prove the biological relevance of the BMP4 in Xenopus embryos can lead to the formation ventralized embryos, i.e. embryos devoid of axial structures, whereas BMP4 depletion can lead to dorsalized embryos, i.e. embryos exhibiting an expansion of dorsal mesoderm [Xmab21l2 is restricted to the primitive eye field, optic recess, optic cup and retina (excluding the choroid fissure), to the tectum and dorsal neural tube, and to branchial arches .To determine whether Xmab21l2 and BMP signaling molecules, we analyzed the effects of isolated Xmab21l2 overexpression. Embryos were injected at the one-cell stage with 800 pg in vitro-transcribed Xmab21l2 alone. We analyzed the phenotype of injected tailbud embryos as well as the expression of dorsal markers at gastrulation. A significant percentage of injected embryos  featured various degrees of dorsalization/anteriorization (DAI 6–8), with a well formed, sometimes enlarged head, a broad cement gland, a short and/or bent axis  with expanded and/or enhanced Chordin gene expression . Our results are in sufficient agreement with those previously obtained by others [Xmab21l2 overexpression produces neither signs of complete dorsalization, such as Janus twins or radial eyes [Prior to addressing the nature of the interaction between s Figure . We preps Figure , suggesty others , confirmXmab21l2 can compensate the effects of BMP4 overexpression, Xenopus embryos were injected at the one-cell stage with in vitro-transcribed BMP4 mRNA alone , or coinjected with Xmab21l2 mRNA . We scored the formation of dorsal structures in tadpole stage embryos by using the dorsoanterior index [BMP4 overexpression (0.6 ng) . Likewise, whilst complete dorsal structures (DAI = 5) , suggesting that Xmab21l2 antagonizes BMP4 in its ability to ventralize embryonic mesoderm in vivo  Figure . Coinjec) Figure , bottom in situ hybridization, we analyzed the expression of molecular markers of dorsal . Both cell- and embryo lysates were subjected to immunoprecipitation, using an anti flag monoclonal antibody, whereas an anti-myc antibody was utilized for immunodetection in Western blotting. Experiments conducted in transfected P19 cells showed that SMAD1 coprecipitates with MAB21L2, and that the interaction is enhanced by activation of BMP signaling, which is required for nuclear localization of receptor-dependent SMADs  [Mab21l2-cotransfected COS7 cells was compared to the baseline reporter activity scored in cells transfected with the GAL4 DBD alone (G-D) or with a transcriptionally inactive GAL4 DBD fusion protein,  [Gal4-Mab21l2-transfected cells only, suggesting that MAB21L2 may function as a transcriptional repressor or co-repressor  , either NT, G-N) . Reporter Figure . The samC. elegans mab-21 undergoes inhibitory regulation by TGF-beta superfamily signals [Mab-21 genes have been isolated in several species and have been shown, through genetic and epigenetic loss-of-function experiments, to play key roles in various developmental processes, ranging from gastrulation and neural tube closure to eye and lens formation ,10,12,13 signals , the molXenopus embryo, and in murine embryonic carcinoma cells, indicate that MAB21L2 interacts functionally with SMAD1, a nuclear transducer of BMP2/4/7 signaling. Overexpression of Mab21l2 complements the effects of BMP4 overexpression, in keeping with the epistatic interactions observed in nematodes [pseudocoelomates appear to be strongly conserved in evolution.Our results, obtained in an anamniotic model system, the ematodes . Thus, gAs the nuclear localization of MAB21L2 suggested, our pull-down and immunoprecipitation results indicate that MAB21L2 is a new interactor of the receptor-activated transducer SMAD1. Whilst the interaction between MAB21L2 and SMAD1 appears to be direct, our results do not seem to favor a direct contact between MAB21L2 and SMAD4.Xenopus laevis and mouse embryos. The increasing levels of MAB21L2 protein observed starting at midblastula transition may be required to compensate mesoderm-ventralizing signals triggered by BMP2/4. In the absence of MAB21L2 genes, completion of gastrulation may be hampered, resulting in various abnormalities of dorsoventral polarity [Mab21l2 downstream genes will make it possible to investigate mab-21 – BMP4 interactions from the opposite standpoint, i.e. by looking at the modulatory effect of BMP signaling on the expression of Mab21l2 targets.The interaction of MAB21L2, a transcriptional repressor, with nuclear transducers of BMP signaling offers a possible explanation for the results of loss-of-function experiments conducted by other investigators in polarity ,12. Resupolarity . Our resMab21l2 in early embryogenesis, the gene is clearly expressed at high levels and in a tightly localized fashion in neurulation and morphogenesis as well, likely contributing to neural tube and eye formation. Indeed, because of redundancy in the mab-21 gene family, many of the early effects of mab-21 gene mutation may be masked, uncovering these genes' roles in later stages of development and postnatal life.Although our results are more directly relevant to understanding the functions of Mab21l1 in lens formation has been recently documented in knock-out mice [mab-21 leads to disruption of the retinal anlage, where the gene is strongly expressed in a territory overlapping with the Bmp4 and Xvent2 expression domain. Recent studies have shown that BMP signaling plays a relevant role in guiding morphogenesis along the dorsoventral axis of the chick [Xenopus [In late developmental processes, well beyond the end of gastrulation, MAB-21 proteins may prolong their dynamic interaction with BMP signaling transducers. As mentioned, a cell-autonomous requirement for out mice  that devhe chick  and Xeno[Xenopus  retina. Mab21l1 and Mab21l2 are prominently expressed in the embryonic midbrain, both dorsally and ventrally, and in prosomere 1 [somere 1 ,6. Rolessomere 1 , and, mosomere 1 .Mab21l2 is expressed mostly in dorsal territories. In cerebellar development, it is expressed in the cerebellar plate and, later on, in granule cell neurons [Mab21l2 at several of those sites, and their developmental roles have been at least partially addressed: as an example, granule cell specification can be induced by BMP7 overexpression in the dorsal and ventral rhombencephalon [Mab21l2 is expressed in a dorsal territory, where BMP2/4 signaling plays a key role in the specification of neuronal identities alongside the dorsoventral axis of the neural tube [Throughout the hindbrain and spinal cord,  neurons . BMP sigcephalon . BMP7 iscephalon . Likewisral tube -34.Mash1 maintains competence for neuronal differentiation [Mab21l2 may thus participate in specifying neuronal and non-neuronal cell fates by interacting with BMP signals.Finally, in postmigratory neural crest, the interplay between BMP2/4 and ntiation . In neurmab-21 gene function in vertebrate systems, demonstrating that MAB21L2 interacts functionally with the BMP4 signalling pathway and physically with its best characterized nuclear transducer, SMAD1. Furthermore, we show that MAB21l2 can act as a powerful transcriptional repressor when targeted to an heterologous promoter. Further work is clearly required to determine if MAB21L2 binds DNA, what its binding specificity is, or if it only exerts its effect in a DNA-binding-independent fashion. More broadly, additional studies are required to clarify the role of mab-21 genes in the context of BMP signaling, and their likely function as a molecular switch linking different regulatory pathways in development and disease.Our results provide a biochemical and molecular foundation for future studies of Images were obtained either by traditional photography (slides 1–3) or by electronic scanning of autoradiography films. All images were processed through the Adobe Photoshop 7.0 software or through the Adobe Illustrator 10 software.Xenopus laevis homolog of mouse Mab21L2 was isolated by screening a Xenopus stage 28–30 lambdaZapII library (Courtesy of Richard Harland) using the murine homolog as a probe. The 2.8 Kb clone isolated contained the full length Xmab21l2 cDNA (GenBank AF040992) as well as 5' and 3' regions. The coding region of Xmab21 was excised using NotI and BalI (1.3 Kb) and subcloned into pBluescript for whole mount in situ hybridization, or BamHI-StuI (1.1 Kb) and subcloned into pT7TS for overexpression in embryos and into pCDNA3 for cell transfection.The Xenopus females were primed 1000 U of human chorionic gonadotropin  the night before collection. Ovulated eggs were fertilized with testis homogenates and allowed to develop in 0.1X MMR . Jelly coats were removed in 3.2 mM DTT, 0.2 M Tris pH 8.8. Embryos were staged according to Nieuwkoop and Faber [All animal experimentation was conducted according to the stipulations of the Institutional Animal Care and Use Committee, San Raffaele Scientific Institute. In order to obtain embryos, nd Faber  and fixend Faber  for in sFor histological examination, wt or injected and stained embryos were fixed in MEMFA, embedded in wax, cut into 10 μm sections, dried onto slides, dewaxed with xylene and dehydrated with alcohol. Sections were rehydrated and stained with an orange G solution  and subsequently with an orange G-aniline blue solution , and dehydrated ,38.Whole-mount in situ hybridization was performed on staged embryos as described by . The antXenopus Mab21l2 protein wastested by Western blot on total extracts from X. laevis embryos. The antibody did not work successfully in IHC or IP experiments, suggesting that it fails to recognize the native epitope.A 14aa C-terminal stretch from the mMab21l2 protein was chosen as immunogenic peptide to obtain antisera in rabbits. Peptide was synthesized, lyophilized and coupled to Keyhole limped hemocyanin (KLH). Coupled peptide was used for immunization of two rabbits. Immune and preimmune serum were controlled by ELISA using the same peptide coated at different serial dilutions. Immune serum was then purified by affinity chromatography: briefly, 20 mg peptide was coupled on CNBr-Sepharose-4B resin (Pharmacia), according to the standard procedure suggested. 50 ml antiserum diluted in 1:1 in 1X PBS was applied on the peptide/CNBr-Sepharose-4B over night at 4°C. After washing the resin with 50 ml PBS and 50 ml 3 M NaCl, bound antibodies were eluted using 100 mM Glycine pH 1.8. Amount, specificity and purity of the antiserum was tested by the Bradford assay, ELISA and SDS-PAGE/Coomassie staining. Purified antibody was tested for specificity in western blot on total protein extracts from cos-7 cells transfected with mMab21l2-myc fusion protein or mock vector, and on total brain extract from mouse embryos. One single band of 41 KDa was detected by anti Mab21l2 antibody. The same 41 kD band was detected by WB in lysates from transfected COS-7 cells, and was missing in untransfected cells. Cross-reactivity of anti mouse Mab21l2 antibody with the Protein concentrations in extracts were quantitated through Bradford assays. Extracts were gel-fractionated by denaturing polyacrylamide gel electropohoresis SDS-PAGE. Gels were transfered onto nitrocellulose filters. Even loading was confirmed by Ponceau staining of nitrocellulose filters. Imunostaining was conducted as described .Xmab21-T7TS was linearized with EcoRI and transcribed with the T7 RNA polimerase, N-MycMab21L2-CS2+ was linearized with Asp718 and transcribed with the SP6 RNA polymerase. The Flag-Smad1 was excised from the human construct Flag-Smad1-CMV5 (Courtesy of J. Massagué) and subcloned into the vector pCS2+. The Flag-Smad1-pCS2+ vector was linearized with Asp718 and transcribed with SP6. BMP4 expression constructs were a kind gift of N. Ueno. Capped mRNA was synthesized using the Ambion Message Machine kit according to manufacturer's instructions. Injections were performed in 4% Ficoll in 1X MMR. Embryos were injected animally at the one-cell or two-cell stage into one or both blastomeres. The amount of mRNA injected is given in the text.The vector COS7 cells were maintained in DMEM Medium (Gibco Brl) supplemented with 10% FBS (Euro Clone), 2 mM L-glutamine (Euro Clone), 100 IU/ml penicillin, and 100 É g/ml streptomycin. Calcium phosphate transfection was performed in a 60 mm Petri dish with different amounts of the expression constructs, 5 É g of reporter plasmids, and 100 ng of pRL-TK (Promega) as an internal control. 18 h after transfection the cells were washed twice with PBS1X and the medium was replaced with DMEM Medium supplemented with 10% FBS, 2 mM L-glutamine (Euro Clone), 100 IU/ml penicillin, and 100 É g/ml streptomycin. Cells were harvested 36 h after the transfection, lysed, and assayed for luciferase activity . All luciferase assays were performed in triplicate.Xenopus laevis lysates were obtained in the same way starting from injected embryos harvested by centrifugation.P19 cells were maintained in MEM alpha medium (Gibco Brl) supplemented with 10% FBS (Euro Clone), 2 mM L-glutamine (Euro Clone), 100 IU/ml penicillin, and 100 μg/ml streptomycin. Calcium phosphate transfection was performed in a 100 mm Petri dish with 10 μg of mycMab21l2 expression construct and 10 μg of flagSmad1 expression construct. 24 h after transfection the medium was replaced with MEM Alpha Medum supplemented with 1% FBS 2 mM L-glutamine (Euro Clone), 100 IU/ and 40 ng/ml BMP4 (R&D Systems). Cells were harvested 36 h after the transfection with TEN Solution  and centrifugated for 15 minutes at 5000 RPM. Cell pellets were frozen at -80°C o/n, thawed at RT and resuspended in 5 volumes of Extraction Buffer . The samples were centrifugated at 34000 RPM for 30 minutes at 4°C and the supernatants were harvested. The concentration of the proteins was determined by BCA assay (Pierce). 3; 0.1% triton), once with TSA solution  and once with 50 mM Tris-Cl pH 6.8. The protein complexes were eluted by addition of Sample buffer  followed by boiling for 5 minutes and separated on an SDS-polyacrylamide gel followed by anti-myc (1 μg/ml of clone n°9E10) immunoblotting. The secondary antibody was a goat anti-mouse antibody HRP-conjugated (1:30000 Bio-Rad). The blots were developed with the Supersignal West Pico reagent (Pierce).After preclearing with 50 μl of protein G-sepharose o/n at 4°C, lysates were incubated with 25 μg of M2 anti-Flag monoclonal antibody (Sigma) and 50 μl of protein G-Sepharose o/n at 4°C. The resin was washed twice with Dilution Buffer  was induced by β-D-thiogalactopyranoside (IPTG)  when the optical density (OD600) reached 0.7–0.9. Cells were harvested after 3 hours, resuspended in lysis buffer , and sonicated. The purification protocol was performed with Ni-NTA Agarose (Quiagen) beads and the elution with 250 mM Imidazole (Sigma).The protocol was based on manufacturer's recommendations for Ni-NTA Agarose (Qiagen). His-ZZ fusion protein expression in bacterial cultures . The beads were recovered by centrifugation and washed ten times with 0.4 M KCl. The beads were incubated 1 h with 250 μg of precleared lysates of P19 treated and untreated with 40 ng/ml of BMP4. The beads were washed 5 times with 0.2 M KCl; the protein complexes were eluted by addition of Sample buffer followed by boiling for 5 minutes, and separated on an SDS-polyacrylamide gel for anti-Smad1 (1:2000 Santa Cruz) immunoblotting. The secondary antibody was a goat anti-rabbit HRP-conjugated antibody (Bio-Rad). The blots were developed with the Supersignal West Pico reagent (Pierce).120 μg of His-ZZ-Smad1 fusion protein was incubated 1 h at 4°C with 25 μl of IgG-Sepharose resin (Amersham). The beads were recovered by centrifugation and washed ten times with 0.4 M KCl.The beads were incubated 1 h with 250 μg of precleared lysates of COS7 cells transfected with mycMab21l2. The beads were washed 5 times with 0.2 M KCl; the protein complexes were eluted by addition of Sample buffer,boiled for 5 minutes and separeted on a SDS-polyacrylamide gel for anti-myc immunoblotting.2; 1 mM DTT; 0.2% NP-40, 10% glycerol). The beads were recovered by centrifugation and washed with Binding buffer. In-vitro translated, 35S-methionine-labeled Smad1 and Smad4 were prepared with the TNT coupled transcription/translation system (Promega). 45 μl of the TNT reaction were mixed with the His-ZZ-Mab21l2 bound to the IgG-Sepharose beads. The mixture was incubated for 2 hours at 4°C in Binding buffer. The Sepharose-protein complex was washed four times with Wash buffer , eluted by addition of Sample buffer followed by boiling for 5 minutes and separated on SDS-polyacrylamide gels. Gels were fixed and stained with 50% methanol, 10% acetic acid and 0.1% Comassie Blue for 20 minutes. After the incubation with Sodium salicylate 1 M (Fluka) for 20 minutes to improve the radioactive signal the gels were dried and exposed to Kodak X-Omat film o/n at -80°C.120 μg of His-ZZ-Mab21l2 fusion protein was incubated 1 h at 4°C with 25 μl of IgG-Sepharose beads (Amersham) in Binding buffer (50 mM Tris pH8; 50 mM KCl; 5 mM MgClin situ hybridizations and in vivo overexpression experiments.DB did AB did pull-downs, immunoprecipitations and luciferase assays.RL designed peptides and produced an anti MAB-21 polyclonal antibody.VZ produced constructs for, and carried out DNA-binding assays and luciferase assays. In addition he helped design and evaluate all cellular assays.GGC designed and coordinated the experimental work.All authors read and approved the final manuscript


A 24-y-old woman was admitted to the emergency department having had a generalized seizure . She was on the sixth day of treatment with 300 mg daily of slow-release bupropion (Zyban SR) as an aid to smoking cessation. She had a past medical history of tonsillectomy and hay fever, for which she was taking budesonide nasal drops . She was on no other medication. There was no history of head trauma, liver disease, or alcohol withdrawal. Clinical examination, including neurological examination, was normal. The patient's weight was 48 kg. Her blood pressure was 130/80 mm Hg. Electrocardiogram showed a sinus tachycardia at 102 beats per minute. Radiography of the skull and a computed tomography scan of the brain without contrast were both normal. The patient's blood glucose, urea, electrolytes, and liver function tests were all normal. Her serum calcium was 2.01 mmol/l  and her hemoglobin was 116 g/l . The bupropion was discontinued, and the patient recovered without any further seizures or other neurological sequelae. Bupropion recently came onto the Mauritian market as an aid to smoking cessation. This case report is a useful reminder to clinicians of the risks of taking the drug Originally developed as an antidepressant, bupropion has more recently been licensed in many countries as an aid to smoking cessation. It came onto the Mauritian market as a smoking cessation aid in November 2003.www.bnf.org) recommends starting the drug one to two weeks before the target smoking stop date, initially at a dose of 150 mg daily for 6 d and then 150 mg twice daily. The maximum period of treatment is 7–9 wk; treatment should be discontinued if abstinence is not achieved by 7 wk.The British National Formulary .Bupropion is associated with a dose-related risk of seizure. The Medicines Control Agency states that the incidence of seizures is one in 1,000 based on doses up to the maximum recommended daily dose of 300 mg per day . The seiUp to 24 July 2002, in the UK there were 184 reports of seizures suspected as being associated with the use of bupropion [In the case we have presented, the patient had no current or previous history of seizure disorders, bulimia, or anorexia nervosa. She was on the sixth day of treatment with bupropion, taking 300 mg per day in two separate doses. She had no other predisposing risk factors for seizure. In particular she was taking no prescription medications, or over-the-counter medications (such as those containing ephedrine products) that are known to lower the seizure threshold. While systemic steroids can lower the seizure threshold, systemic absorption of the nasal steroid drops the patient was taking is an extremely unlikely cause for her seizure (she was not taking a high dose of high-strength drops). Her weight (48 kg) may have been an important factor, since bupropion reaches higher plasma levels in smaller individuals—indeed clinical trials of the drug have generally excluded patients weighing under 100 lb (about 45 kg) .This case report is a useful reminder to clinicians that bupropion is associated with a dose-dependent risk of seizures.In about half of the reports of seizure associated with bupropion, there was a past history of seizures and/or risk factors for their occurrence.It is extremely important to adequately assess seizure risk before prescribing bupropion.Patients should be made aware of the possible adverse effects of the drug.


This study investigated a) the amount and types of out-of-pocket expenditures by patients for nominally free services in a large public hospital in Bangladesh, b) the factors influencing these expenses, and c) the impact of these expenses on household income.Eighty-one maternity patients were interviewed during their hospitalization in the Dhaka Medical College Hospital. Patients were selected by quota sample to match the distribution of maternity patient categories in the hospital. Patients were interviewed with a semi-structured, in-depth questionnaire.All interviewees incurred substantial out-of-pocket expenditures for travel, hospital admission fees, medicine, tests, food, and tips. Only two of the expenditures, travel expenses and admission fees, were not supposed to be provided free of charge by the hospital. The median total per-patient expenditure was $65 (range $2–$350), equivalent to 7% (range 0.04%–225%) of annual household income. Half of all patients reported that their families had to borrow to pay for care at interest rates of 5%–30% per month. A third of these families reported selling jewelry, land or household items to moneylenders. The rural patients reported more difficulty in paying for care than the urban patients. Factors increasing the expenditures were duration of hospitalization, rural residence, and necessary  and unnecessary (e.g. episiotomy) medical procedures.Free maternity services in Bangladesh impose large out-of-pocket expenditures on patients. Authorities could reduce the burden by reducing the duration of hospital stays, limiting use of medical procedures, eliminating tips, and moving routine services closer to potential users. Fee for service could reduce unofficial expenditures if the fee were lower than and replaced typical unofficial expenditures, otherwise adding service fees without reform of current hospital practices would lead to even more burdensome expenditures and inequities. In developing countries governments often subsidize services at public health care facilities and provide them free of charge to users. However, evidence suggests that users still incur large expenditures using the 'free' services for such things that are supposedly provided without charge. Studies have found that patients incurred substantial out-of-pocket expenditures for medicine, food and travel from the use of 'free' public health facilities -3. A stuThree previous studies have explored issues related to patient expenditures in Bangladesh ,10,11. NThus, this study examined the type, amount and household financial results of out-of-pocket expenditures by patients for nominally free services in a large government hospital in Dhaka. The study also identified the factors and medical practices producing and influencing the out-of-pocket expenditures. Plans to begin fees for service in Bangladesh make it important to document the amount of money actually being paid by the patients under the present system. If current expenditures are large, fee for service may have serious negative impacts on utilization and on the economic well-being of Bangladeshi households. If current expenditures are modest, it is possible that such fees will have a lesser impact.The study was conducted in the Department of Obstetrics and Gynaecology (ObGyn) of the Dhaka Medical College Hospital (DMCH). DMCH is the largest teaching hospital in Bangladesh with 850 beds located in the capital city. DMCH is government funded and provides a wide range of out- and in-patient services. Public hospitals have two payment categories for in-patients: non-paying and paying. Patients first go to an out-patient unit for diagnosis where they are categorized as out- or in-patient. Those categorized as in-patient are then classified as paying or non-paying by observing the clothes and general appearance of the woman and any accompanying relatives. Non-paying patients pay only the hospital admission fee. Paying in-patients are charged the fees for hospital admission, bed, and surgery. The various fees are: hospital admission fee: $0.23, bed fee: $1.34–3.50 per day, surgery fee: $12.50–125. Taka was converted into US dollars using the 1994 exchange rate of US$1.00 = Taka 40.00. Neither patient category is supposed to pay for medicine, tests, food, nursing and other support services during hospitalization; these commodities and services are theoretically provided free by the hospital.The study interviewed 81 non-paying in-patients hospitalized for reproductive health conditions (about two thirds were for maternity conditions). Patients were selected by quota sample matching the distribution of the patient categories in the hospital i.e. the selected medical conditions accounted for the greatest number of ObGyn admissions reported for the hospital, and also reflect the causes associated with high maternal mortality and morbidity in Bangladesh . These iInformation was collected on various characteristics of the study participants. Demographic characteristics included age, education, marital status, and residence. Socio-economic characteristics included occupation and annual household income. Information was also collected on underlying medical condition. Out-of-pocket expenditure related information included types and amounts of expenses incurred during hospitalization such as those for travel, medicine, food, fees, etc. Factors influencing expenditures included type of treatment received and duration of hospitalization. Sources of funds included amount borrowed and interest charged for borrowed amount.Data were collected from patients and their relatives with semi-structured open-ended questionnaires between January – June 1994. The interviewers were physicians employed in DMCH. The interviewers selected the cases by diagnosis from patient admission records. To minimize possible selection bias the first case was selected randomly from the records and then every third case was selected. The selected patients were interviewed a minimum of three times to minimize recall error. Recall error was also minimized as information was collected while patients were still hospitalized. During the first interview demographic and socio-economic information was collected with structured questions. During the second and third interviews information related to expenditures was collected with open-ended questions.To illustrate the data collection process a description of an interview with a typical C-section patient follows. C-section patients are usually hospitalized for two weeks in DMCH. On the first day of hospitalization an interviewer collected information on patient's age, education, marital status, etc. On the eighth day of hospitalization the second interview collected information on treatment received, treatment related out-of pocket expenditures, annual household income, amount of money borrowed to pay for treatment, source of borrowed money, and interest rate charged. On the fourteenth day the third interview collected more monetary information on out-of-pocket expenditures, and on expected expenditures immediately after leaving the hospital. This survey did not cover the expenditures for the full course of the treatment. Expenditure estimates were derived for the duration of the current hospitalization only, i.e. from the day of admission until the day of discharge. Expenditures immediately before admission and after discharge from the hospital included only travel expenses to and from the hospital for the patient and her accompanying relatives.The median age of the study participants was 26 years (range 15–60 years). The majority (88%) of the patients were married, the rest were separated (4%), divorced (2%), and unmarried (1%). Forty-four percent of the patients lived in rural areas. The median annual household income was $750 (range $3–$6000) per respondent. The annual household income was higher for the urban  than the rural  respondents.All 81 patients interviewed reported incurring substantial out-of-pocket expenditures during their hospitalization. These out-of-pocket expenditures were for travel, hospital admission fee, medicine, tests, food, tips, and other items. As expected there were expenditures related to travel and admission fees which the hospital is not supposed to subsidize. But there were also expenditures for medicine, tests, food, tips, and other items which were supposed to be provided free from the hospital but were not.The median total expenditure for hospitalization was $65 range $2.15–$350) per patient. On average, 61% of these expenditures ($49) were for services and commodities that were supposed to be provided free from the hospital but were not. The per patient median expenditure for the various expense categories were: medicine $26, tests 0, tips $1.25, food $1.25, other items $4.38, travel $22.25, and hospital admission fee $0.25. On average, medicine constituted 42%, travel 38%, tests 5%, food 4%, tips 2%, admission fees <1%, and others 8% of the total expenditures. C-section and hysterectomy cases had the highest median expenditures. Table .15–$350 All patients were supposed to be provided required medicines free from the hospital but were not. Medicines included antibiotics, analgesics, syringe, catheter, blood, and so forth. Medicine was usually bought when patients were admitted at night. The medicine required for treatment is ordered by the on-duty physician but it takes several hours for the hospital management to process the order. Thus, no free medicine is available immediately. To start the treatment, the on-duty physician requests the patient's relatives to buy the medicine which is purchased from nearby private pharmacies.All tests  are supposed to be provided by the hospital but sometimes the patients had the tests done in a private laboratory because waiting time for tests is very long in the DMCH due to the high patient load.ayahs (cleaning ladies) or nurses to provide necessary services. Thus, food was usually bought from a vendor or brought from home for both patient and relatives.Food is provided by the hospital but the interviewees found the hospital food of poor quality or totally lacking . Relatives usually stayed with the patient in the hospital because of lack of bakshish) are payments made to ayahs and guards. Ayahs were given tips for routine services such as pushing the patient's trolley to and from the labour/operation room, shaving the patient before delivery/surgery, giving enemas, etc. Guards at the gates were tipped each time a relative came to visit the patient during non-visitor hours. However, ayahs and guards are salaried hospital employees and are supposed to provide these services free of charge. The patients were reluctant when talking about the tips probably because they were still hospitalized and depended on these employees for access to certain services.Tips  and the newborn baby (e.g. blanket) that were supposed to be provided by the hospital free of charge but were not.Travel expenses are not supposed to be provided by the hospital. Travel expenses consisted of travel to and from the hospital by the patient and any accompanying relatives, and travel expenditures of relatives during hospitalization for purchasing medicine and food for the patient. The patients came to DMCH because they expected 'free' and 'affordable' services compared to private clinics, or they were referred from a primary/secondary level facility, or to get better treatment here. Patients from rural/peri-urban areas took longer to reach DMCH than those from urban Dhaka .This expense is also not supposed to be covered by the hospital. The official price of admission was $0.23, but it was zero for two patients and more than the official price for half of the patients interviewed. The study could not elicit the reason the patients paid more than the official price. When probed the patients could not or would not elaborate beyond the amount paid. The patients were very reluctant when talking about paying more than the official price for the admission fee.Duration of hospitalization and rural residence of the patients increased the out-of-pocket expenditures. Rural residence increased the travel expenses and thus the total expenditures. Longer duration of hospitalization increased virtually all expenditures. The median duration of hospitalization was 8 days (range 1–34 days) per patient. Duration of hospitalization was the longest for hysterectomies followed by C-sections. Duration of hospitalization was related to severity of medical condition (e.g. eclampsia), necessary medical procedures (e.g. hysterectomy), and unnecessary medical procedures (e.g. episiotomy). One day of extra hospitalization increased expenditures by $2.30 per patient.medical procedures increased the patient expenditures. Episiotomy increased expenditures as patients were hospitalized for a longer duration and resulted in the purchase of more medicine. Episiotomy increased expenditures for both uncomplicated NVD (by 37%) and eclamptic NVD (by 84%) compared to cases where no episiotomy was performed (data not shown). The medical reason for performing episiotomies is the prevention of perineal tearing but because of a high case load at DMCH physicians perform episiotomies to reduce the length of delivery time, effectively turning hospital expenditures into patient expenditures. Eclampsia increased the expenditures for NVD by 180% (data not shown). Eclampsia is not under the control of the health system or the patient, and procedures used for treating eclampsia are unavoidable.Choice of C-sections caused higher patient expenditures compared to NVDs (median $119 and $63 respectively) because C-sections had a longer duration of hospitalization and required more medicine. Elective C-sections and eclamptic C-sections incurred similar expenses because elective C-sections were hospitalized for a longer duration even though there were no complications. Vaginal hysterectomies were 25% less expensive than abdominal hysterectomies because they required less invasive procedures, used local anaesthesia, and had a shorter duration of hospitalization.The respondents said that they were willing to pay for care. However, rural households reported more difficulty in paying for care than urban households. Difficulty was inferred from the number of households who borrowed to pay for care, and the ratio of the amount borrowed to the annual household income. The median patient expenditure was equivalent to 7% (range 0.04%–225%) of annual household income, and was higher for rural  than urban  respondents. Half (n = 40) of the households reported borrowing to pay for care. The patient who spent 225% of her annual household income was a rural patient who had a hysterectomy for prolapsed uterus. Surgical patients like her are usually hospitalized for a month as they require more tests than non-surgical patients. This patient's total expenditures were not higher than the others who also had a hysterectomy, however, her annual household income was much lower than that of the others.More urban n = 23) households borrowed than rural (n = 17) households but the amount borrowed was higher for rural households. The median amount borrowed per household was $38 and was equivalent to 8% (range 0.58%–208%) of the annual household income. On average, the rural households  borrowed almost double the amount than the urban households . Most often (n = 30) money was borrowed from friends and relatives without interest. When borrowing was from money-lenders (n = 10), households reported interest rates of 5%–30% per month. Three households put up security such as jewelry, land and household goods when borrowing money from moneylenders. The highest reported percentage of money borrowed to income for a rural household was 208% compared to 28% for an urban household. Finally, greater amounts of money were borrowed by C-section and hysterectomy patients than the other categories of patients. Table  householThe study findings indicate that all the surveyed patients incurred substantial out-of-pocket expenditures for a one time hospitalization. The median per patient expenditure was $65, and two-thirds of these expenditures were for commodities and services that were supposed to be provided free by the hospital but were not. Half the households borrowed to pay for care since they did not have the ability to pay . A third of these households sold jewelry, land or household items to moneylenders. The rural households reported more difficulty in paying for care than the urban households. The rural patients had lower income but incurred higher expenditures and borrowed larger amounts than the urban patients.The study data are a decade old but worth presenting because Bangladesh is only recently beginning health sector reform and fee for service. Also, the hospital practices with regard to providing maternity care remain the same, in the hospital studied and in other public hospitals in Bangladesh. Other limitations of the study are its small sample size and that all the interviewees came from only one hospital. The small sample size is the norm when doing in-depth interviews. When a paper is qualitative not quantitative no statistical tests are customarily done. The results are striking despite the limitations. The costs related to NVD and C-section were much higher in this study than that estimated by Nahar et al. . Possible reasons for the differences are: a) recall error higher in the Nahar et al. study as they interviewed post-partum mothers, whereas, this study interviewed patients during hospitalization; b) the Nahar et al. study included only uncomplicated cases, whereas, this study included both uncomplicated and complicated cases. The annual household income was lower in this study compared to the Nahar et al. study ($750 and $1476 respectively). This may be attributable to having rural patients in this study whose income is much lower than urban patients, whereas, Nahar et al. studied only urban patients.Changing current practices regarding the length of hospitalization and medical procedures (e.g. episiotomy) will reduce patient expenditures. This can be achieved by having shorter duration of hospitalization for elective C-section and elective hysterectomy, and limiting the use of episiotomy. Limiting these practices may also lead to lower provider expenditures. Medicine constituted almost half of the total patient expenditures. However, lack of access to medicine resulted from an inefficient management system not from unavailability. Taking less time to process orders would make medicine available quicker to patients and so reduce their expenditures.Hospital management needs to ensure that patients pay only the official rate of admission fees. Hospital management also needs to ensure that patients do not pay tips to salaried hospital staff for routine services. The hospital could arrange for liquid food and hot water for surgical patients in addition to the other services it already provides, i.e. provide a more comprehensive hotel service. Alternatively the hospital could subcontract out its hotel services to those employees who extract tips from patients to provide such services. This may act as an incentive for eliminating the unofficial fees from tips. Making some services such as eclampsia and hysterectomy available at secondary level facilities will benefit the rural patients by reducing the travel expenses. Government hospitals can generate revenue by introducing or increasing some fees for medicine and tests for the paying category of patients but clearly exempting the poorest. How would the system determine who was the poorest? One option would be to let all rural women use hospital for free and have all urban women pay some fee.For service fees not to become a serious barrier to use current systems will have to eliminate or reduce practices that already result in high user expenditures from unofficial payments. Otherwise the added fees may lead to more borrowing from moneylenders, putting lands and goods at risk, and potential impoverishment of more households. A fee for service system needs to be based on information, and more than just setting a price. This can be facilitated by knowing what the patients were already spending in the current system and who were the most at risk of impoverishment. The challenge is to focus on realistic, short term changes that can reduce patient expenditures and inequities. Most of the recommendations of this study depend more on the will of the physicians and the hospital administrators than on the infusion of resources per se.Free maternity services in Bangladesh impose large out-of-pocket expenditures on patients. Authorities could reduce the burden by reducing the duration of hospital stays, limiting use of medical procedures, eliminating tips, and moving routine services closer to potential users. Fee for service could reduce unofficial expenditures if the fee were lower than and replaced typical unofficial expenditures, otherwise adding service fees without reform of current hospital practices would lead to even more burdensome expenditures and inequities.The author(s) declare that they have no competing interests.


Cost utility analysis (CUA) using SF-36/SF-12 data has been facilitated by the development of several preference-based algorithms. The purpose of this study was to illustrate how decision-making could be affected by the choice of preference-based algorithms for the SF-36 and SF-12, and provide some guidance on selecting an appropriate algorithm.Two sets of data were used: (1) a clinical trial of adult asthma patients; and (2) a longitudinal study of post-stroke patients. Incremental costs were assumed to be $2000 per year over standard treatment, and QALY gains realized over a 1-year period. Ten published algorithms were identified, denoted by first author: Brazier (SF-36), Brazier (SF-12), Shmueli, Fryback, Lundberg, Nichol, Franks , and Lawrence. Incremental cost-utility ratios (ICURs) for each algorithm, stated in dollars per quality-adjusted life year ($/QALY), were ranked and compared between datasets.In the asthma patients, estimated ICURs ranged from Lawrence's SF-12 algorithm at $30,769/QALY  to Brazier's SF-36 algorithm at $63,492/QALY . ICURs for the stroke cohort varied slightly more dramatically. The MEPS-based algorithm by Franks et al. provided the lowest ICUR at $27,972/QALY . The Fryback and Shmueli algorithms provided ICURs that were greater than $50,000/QALY and did not have confidence intervals that overlapped with most of the other algorithms. The ICUR-based ranking of algorithms was strongly correlated between the asthma and stroke datasets (r = 0.60).SF-36/SF-12 preference-based algorithms produced a wide range of ICURs that could potentially lead to different reimbursement decisions. Brazier's SF-36 and SF-12 algorithms have a strong methodological and theoretical basis and tended to generate relatively higher ICUR estimates, considerations that support a preference for these algorithms over the alternatives. The "second-generation" algorithms developed from scores mapped from other indirect preference-based measures tended to generate lower ICURs that would promote greater adoption of new technology. There remains a need for an SF-36/SF-12 preference-based algorithm based on the US general population that has strong theoretical and methodological foundations. Health-related quality of life (HRQL) measures have many applications, including the measurement of population health status and outcomes of medical interventions that subsequently can be applied to economic evaluations of health care interventions. One such method of economic evaluation, cost utility analysis (CUA), is a special form of cost effectiveness analysis that evaluates incremental costs and effects of an intervention by assessing health effects using quality-adjusted life years (QALYs) . QALYs iOptimally, CUA is used to guide the allocation of resources on a societal level. The Panel on Cost Effectiveness in Health and Medicine recommended that community preferences for health states collected from a representative sample of the US general population should be "the most appropriate ones for use in a Reference Case analysis" for US decision makers . Such anAn important development in health services research has been the emergence of algorithms that generate single preference-based summary scores based on items, domain scores, or summary scores from the Short Form 36 (SF-36)  and SF-1The purpose of this study was to examine how choice of algorithm for the SF-36/SF-12 might affect decision-making. The specific objectives for the study were to calculate ICURs by applying each algorithm to data from 2 different studies that included longitudinal assessments of the SF-36, to compare the ranking of each algorithm-based ICUR across conditions, and finally to interpret whether differences in ICURs generated by each algorithm had the potential to affect decision making. There were two specific hypotheses. First, ICURs calculated from different algorithms were expected to differ because preferences derived from those algorithms had been found to be different . Second,To illustrate the outcomes of CUA using the different SF-36 algorithms, data with empiric responses to the SF-36 from patients were used from two different sources and conditions: (1) a clinical trial of adults with asthma ; and (2)The second source of data was a natural history of HRQL after stroke. Stroke patients who were hospitalized with a confirmed ischemic stroke and consented to participate were included. Patients were excluded if they were ≤ 18 years old, could not comprehend English-based questionnaire, lived > 150 kilometers from Edmonton, Alberta, had hemorrhagic or lower brain stem stroke, coma, global or Wernicke's aphasia, or life expectancy was less than 6 months for any medical reason. Patients were enrolled in the study within two weeks of stroke and no later than 3 weeks after stroke. Health status measures, including the SF-36, were self-assessed by patients. For this analysis, responses to the SF-36 at baseline and 6 months were used. Both the stroke and asthma studies used version 1 of the SF-36.The SF-36 has been traditionally described as a psychometrically-derived generic health status profile, with 8 subscales and two summary scores, the physical component summary (PCS-36) score and the mental component summary (MCS-36) score. The eight domains include physical functioning (PF), role limitations-physical (RP), bodily pain (BP), general health (GH), mental health (MH), role limitations-emotional (RE), vitality (VT), and social functioning (SF). The SF-12 is a shorter, 12-item version of the SF-36 that does not generate domain scores but provides summary scores, the PCS-12 and MCS-12, that are highly predictive of the PCS-36 and MCS-36 . Scores st generation if the algorithm was derived from directly elicited preferences, and denoted as 2nd generation mapping if the SF-12/SF-36 algorithm was based upon scores from an indirect preference-based HRQL measure, such as the EQ-5D. Note that these algorithms relate to the most recently advocated algorithms, as several authors published earlier algorithms and subsequently published updates (e.g. Shmueli) [Nine publications that derived 10 unique preference-based algorithms for the SF-36 or SF-12 were identified (Table Shmueli) . For breBrazier and colleagues constructed an econometric model for predicting health state valuations by first revising the SF-36 into a health status measure with 6 domains called the SF-6D . Using aFryback and colleagues predicted Quality of Well-being Index (QWB) scores from SF-36 domain scores using data collected from the Beaver Dam Health Outcomes Study . A six-vNichol and colleagues mapped the SF-36 to the preference-based Health Utility Index Mark 2 (HUI2). They estimated HUI2 scores from SF-36 domain scores and sociodemographic variables from a sample of Southern California Kaiser Permanente members . The NicShmueli updated an examination of the relationship between Visual Analog Scale (VAS) ratings and SF-36 domains provided in a population health survey in Israel by predicting VAS values from SF-36 domains using linear and non-linear regression models . The modFranks and colleagues mapped the SF-12 to the EQ-5D Index and HUI3 using a convenience sample of 240 low-income, predominantly Latino and black patients visiting a community health center in New York . Two equFinally, Lundberg and colleagues investigated the relationship of preference-based measures and the SF-12 based on self-assessed HRQL from a random sample of residents in Uppsala County of Sweden . Linear Empiric data for stroke and asthma were used to ensure that actual health state changes were represented. The present analysis was based on patients who completed both pre- and post- assessments and had no missing items. After the scoring algorithms were applied to SF-36 responses using the 10 algorithms , the chat-tests. The rank order of the ICURs was compared between the asthma and stroke groups using Spearman's correlation coefficient (rs). P-values < 0.05 were considered statistically significant.The pre/post domain and preference-based scores were described for both study groups visit using means and standard deviations. The 95% confidence intervals (CIs) for ICURs were based on the CIs for the preference scores. The pre/post change scores were evaluated with paired Of the 304 patients enrolled in the asthma study, 220 (72.4%) completed both the baseline and final SF-36 assessment. The stroke study had 81 of 124 initial respondents (65.3%) complete the SF-36 at baseline and final follow-up. In comparison to the patients in the asthma study, patients in the stroke study were older (mean age 67.4 years vs. 39.1 years) and had much lower mean average PF, RP, SF, and PCS scores  Table . In the s = 0.60 .Table The development of preference-based algorithms for the SF-36 and SF-12 to facilitate CUA has fostered studies that recognized these preference-based scores can differ from each other and from directly elicited valuations in patients with asthma, hypertension, lung transplantation, and osteoporosis ,20,29,30Although guidelines or thresholds for decision making based on cost per QALY are contentious, cost-effectiveness thresholds that health care decision makers are willing to accept in health care reimbursement decisions exist, if not explicitly, then implicitly. Some guidance has been published. The National Institute of Clinical Effectiveness (NICE) in the UK has indicated they do not have an explicit threshold , while aThe CUA simulations illustrated how selection of a specific algorithm could lead to a different interpretation of the cost-effectiveness of an intervention. In the asthma cohort, algorithms by Lawrence , and theChanges in the rank order of algorithms between conditions can be explained, not only by differences in the preference-based weighting assigned to each of the domain/summary scores, but several additional factors. There are differences in the SF-36 items or subscales retained by some of the methods. For instance, Brazier's SF-6D based on the SF-36 does not include GH, while the score generated by the Shmueli algorithm is largely influenced by the GH domain. The responsiveness/sensitivity of algorithms appears to be somewhat related to the scale range. It was not surprising that Fryback's method produced relatively larger ICURs that implied the intervention was less cost effective, as Fryback's method had a much smaller range of scale relative to the other algorithms Table . AlgoritIn order to provide some guidance in the selection of preference-based algorithms for the SF-36 and SF-12 the algorithms were appraised in the context of their theoretical and methodological foundations, source of community-based preferences, and their relative potential to enhance or deter the uptake of new technology. The study results clearly illustrated that choice of algorithm can affect the estimated ICUR, and that there was a tendency for the Fryback and Shmueli methods to generate higher estimates of ICURs relative to the other algorithms. From a third-party payer perspective, algorithms generating higher ICURs would appeal to third-party reimbursement decision makers with short-term budget constraints, as algorithms that generate higher ICURs provide less encouraging evidence in the adoption of new technology when considered in the context of the $/QALY benchmarks previously discussed. The Panel on Cost Effectiveness of Health and Medicine . InteresA further consideration is the theoretical foundation for the elicitation technique used in the valuation study. Only Brazier employed methodology using the SG technique for first-generation mapping. The SG has the most appeal in economic theory due to its foundations in Expected Utility Theory (EUT), although it has been suggested that the axioms of EUT are empirically flawed , and reqGiven that HRQL may be valued differently between countries , an algoAmong the algorithms presently available, Brazier's algorithms for the SF-12 and SF-36 appear to be most favorable because of their methodological and theoretical basis. From the perspective of the third party reimbursement decision maker, the Brazier algorithms are not among those that tend to encourage adoption of new technology, tending to provide relatively higher estimates of ICUR. For those decision-makers, those algorithms would appear to be more fiscally conservative in the sense that they would not promote the adoption new technology any more than the other methods according to the results of this study. Although similar estimates are obtained using the Brazier SF-36 and SF-12 algorithms , it woulNote that there are several limitations and assumptions to the CUAs simulated in this paper. The primary purpose was to determine whether choice of preference-based algorithm applied to SF-36/SF-12 data has the potential to change the conclusion of a CUA; hence, aspects of the CUA not central to the purpose were simplified. For example, incremental costs were assumed to be constant, whereas in reality, considerable cost variance would be observed across patients. CUAs were performed in two patient populations, i.e., asthma patients and stroke patients, rather than using a single data set, to enhance the generalizability of the results. The rank order of the algorithms is limited to the datasets examined in this study, however, and comparisons of the algorithms across more diseases/conditions and persons with different demographic characteristics may provide stronger evidence of the rank order "stability". Change scores and thus ICUR estimates depend on baseline health status and the impact of an intervention on the various domains of health as captured by changes in responses to the SF-12/SF-36 items. Note that baseline domain scores for asthma and stroke cohorts were lower than US-based population norms. Several algorithm developers provide caveats for the application of their algorithm, including concerns about profiles severely limited by ceiling or floor effects . For insIn summary, SF-36/SF-12 preference-based algorithms tend to generate a wide range of ICURs that can potentially lead to different reimbursement decisions. Brazier's algorithms for the SF-36 and SF-12 had an arguably stronger methodological and theoretical basis, and tended to produce higher ICURs. For decision-makers who consider cost-effectiveness in the decision to reimburse for a new medical technology, selection of an algorithm that generates relatively higher ICURs would provide less convincing evidence of the cost-effectiveness of a new technology and consequently, its uptake. The "second-generation" algorithms mapped from other indirect preference-based measures tended to produce lower ICURs. When an alternative algorithm is selected, sensitivity analysis is recommended using the Brazier SF-12/SF-36 algorithm in order to examine the robustness of CUA. There remains a need for an SF-36/SF-12 algorithm developed from U.S.-based general population preferences with strong methodological and theoretical foundations.Drs. Pickard and Lee were responsible for the conception of the study and acquisition of data. Mr. Wang analyzed the data. Dr. Pickard and Mr. Wang were involved in the drafting of the article. All authors contributed to the interpretation of results and revising the article for important intellectual content.


Astrocytoma is a common aggressive intracranial tumor and presents a formidable challenge in the clinic. Association of altered DNA methylation patterns of the promoter CpG islands with the expression profile of  cancer-related genes, has been found in many human tumors. Therefore, DNA methylation status as such may serve as an epigenetic biomarker for both diagnosis and prognosis of human tumors, including astrocytoma.We used the methylation specific PCR in conjunction with sequencing verification to establish the methylation profile of the promoter CpG island of thirty four genes in astrocytoma tissues from fifty three patients . In addition, compatible tissues  from three non-astrocytoma patients were included as the control.ARF, p15INK4b, p27KIP1, p57KIP2, RASSF1C, RB1, SURVIVINABL, APC, APAF1, BRCA1, CSPG2, DAPK1, hMLH1, LKB1, PTEN, p14, and VHL) displayed a uniformly unmethylated pattern in all the astrocytoma and non-astrocytoma tissues examined. However, the MAGEA1 gene that was inactivated and hypermethylated in non-astrocytoma tissues, was partially demethylated in 24.5% of the astrocytoma tissues . Of the astrocytoma associated hypermethylated genes, the methylation pattern of the CDH13, cyclin a1, DBCCR1, EPO, MYOD1, and p16INK4a genes changed in no more than 5.66% (3/53) of astrocytoma tissues compared to non-astrocytoma controls, while the RASSF1A, p73, AR, MGMT, CDH1, OCT6,, MT1A, WT1, and IRF7 genes were more frequently hypermethylated in 69.8%, 47.2%, 41.5%, 35.8%, 32%, 30.2%, 30.2%, 30.2% and 26.4% of astrocytoma tissues, respectively. Demethylation mediated inducible expression of the CDH13, MAGEA1, MGMT, p73 and RASSF1A genes was established in an astrocytoma cell line (U251), demonstrating that expression of these genes is likely regulated by DNA methylation. AR gene hypermethylation was found exclusively in female patients , while the IRF7 gene hypermethylation preferentially occurred in the male counterparts . Applying the mathematic method "the Discovery of Association Rules", we have identified groups consisting of up to three genes that more likely display the altered methylation patterns in concert in astrocytoma.Seventeen genes (Of the thirty four genes examined, sixteen genes exhibited astrocytoma associated changes in the methylation profile. In addition to the possible pathological significance, the established concordant methylation profiles of the subsets consisting of two to three target genes may provide useful clues to the development of the useful prognostic as well as diagnostic assays for astrocytoma. EGFR) [INK4A initiated cell-cycle arrest pathway (the p16INK4a) [p14ARF [RB1 [the p53 [CD44, integrin, and receptors for various growth factors), and the PTEN genes [Diffusely infiltrating astrocytoma is a leading group of the primary central nervous system tumors, accounting for more than 60% of all primary brain tumors ,2. It maEGFR) , the mai [p14ARF , the RB1[the p53 ), a wideEN genes .de novo DNA methylation in the early development of high eukaryotes. DNA methylation patterns in somatic cells are established during the early development and contribute to the allele-specific transcription silencing of the imprinted genes, including the silenced alleles in the X-chromosome and other chromosomes. The epigenetic pattern (the DNA methylation profiles of the genome) in high eukaryotes is integral to the normal execution of the biological activities in cells and needs to be actively maintained. In addition to the changes linked to the cell lineage specific pattern of gene expression, both global hypomethylation and local hypermethylation of the CpG islands occur progressively as cell ages.Transcription in eukaryotes is regulated at multiple levels and inversely correlated with the hypermethylated state as well as the chromatin condensation. It has been well established that the methylation status of CpG islands directly affects the DNA-protein interactions by eliminating the otherwise occurring sequence specific binding of the transcription factors whereas inducing the DNA-bindings of members of the methyl-CpG binding protein family (MBD). Histone modifications (deacetylation and methylation) may occur subsequently leading to chromatin condensation and a long-term transcriptional silencing status of the affected DNA segments. Over 40% of the protein coding genes have at least one CpG island within or near to their promoter, an strong indication for transcription of which is likely to be under the control of DNA methylation status. Three DNA methyl transferases are involved in the control of the methylation state of the CpGs in genome. DNA methyl transferase I is mainly responsible for the maintenance of the methylation status of the genome after DNA replication, while IIIA and IIIB act principally in the p16INK4a gene that has been frequently hypermethylated in almost all types of the tumors examined [SURVIVIN, has also been reported to be demethylated in human ovarian cancer [Aberrant DNA methylation pattern changes gene transcription that has been etiologically linked to cancer formation ,10. The examined -17 incluexamined . The losexamined . On the examined . A promiexamined , correlaexamined ,22. The n cancer . Despite. However, as the majority of studies had only targeted one or a few genes in rather small patient groups, the concurrent hypermethylation behavior of multiple genes has only been addressed in a limited number of tumor types, such as colorectal cancer. The majority, if not all, of the previous studies on the astrocytoma associated changes in methylation profiles have only examined a small number of genes for methylation status at the promoter CpG island [Methylation profiling of the promoter CpG islands has been an important information gathering process for new insights into our understanding of the role of DNA methylation in both initiation and progression of human carcinogenesis. It would result in development of the DNA methylation based assays for cancer diagnosis as well as identification of the cancer genes suffering from the epigenetic defects G island ,26. In tARF, p15INK4b, RASSF1A, RB1 APC, BRCA1, CDH1, DAPK1, hMLH1, p14and VHL) or designed according to the same principle with assistance of the software packages for the CpG islands identification  and the primer design  [All the experiments were performed according to protocols described previously . The pri design  .With the informed consent of all patients and approval of the ethics committee, the tumor samples were collected from astrocytoma patients (n = 53) during operation at the Tiantan Neurosurgical Hospital in Beijing. The pathological classification of tumor tissues was carried out and the stage of each astrocytoma patients was determined according to the WHO classification . No sign260 nm readings.Total genomic DNA was extracted from frozen tissue specimens (50 – 100 mg) according to a standard protocol with some modifications ,27. Froz4OAC and 300 μl ethanol. After washing with 70% ethanol, the gently dried DNA pellet was dissolved with 30 μl TE at 65°C for 10 min. The DNA sample was finally stored at -20°C until further use. PCR reaction was carried out in a volume of 15 μl with 50 ng or less template DNA with FastStart Taq polymerase  as follows. After an initial heat denaturing step 4 minutes treatment at 94°C, 30 cycles of 92°C for 15 sec, varying temperatures with primer pairs , and only the verified targets were included for the study of the astrocytoma tissues.To optimize the MSP procedure, the M. Sss I treated DNA was used as the methylated control template. In detail, the DNA from a normal liver tissue of the healthy liver donor ,24 was b, where both Pearsong's Chi-square test with Upton's adjustment and Fisher exact test  were used to examine the tissue samples with the low expected values. The relative frequency with a 95% confidence interval (P < 0.05) for a binomial distribution was calculated for the whole as well as each subtype of astrocytoma patients.The methylation data were dichotomized as 1 for the co-existence of the methylated and unmethylated alleles, 2 for methylated allele only and 0 for the unmethylated for both alleles to facilitate statistical analysis using contingency tables. The methylation profiles of each individual gene (in percentage) classified by the genders and grading of the patients were presented both in table and in plot. The statistic analyses for the association between the methylation profile of the gene and each of the clinical-pathological parameters were carried out with the statistics package The concordant methylation behavior of the genes was established by comparing frequency of co-occurrence of 2 to 3 target subsets with a mathematic method, namely Discovery of Association Rules , which i2 atmosphere. When cell culture reached 50% confluence, they were treated with 5-Aza-2'-deoxycytidine (Sigma A3656) at the final concentration 10 and 20 nM, respectively for 3 days. The primer pairs for the RT-PCR , and cDNA was obtained using the Supertranscript plus reverse transcriptase with the oligo-dT as primers. PCR with single pair of the target primers run for 15 cycles, followed by another 15 cycle PCR reactions in the presence of the beta-actin primers (Table U251 cells (an established glioma cell line) were cultured in DEME plus 10% new born calf serum at 37°C in a 5% COTo establish the methylation profile of thirty four genes during the process of astrocytoma development, we recruited 53 astrocytoma patients  for this study. 14 cases were pathologically classified at the Grade I pilocytic astrocytoma , 15 cases at the Grade II diffuse astrocytoma , 12 cases at the Grade III anaplastic astrocytoma , and 12 cases (including 4 recurrent cases) at the Grade IV glioblastoma   as template would be an ideal positive control for the absence of methylated targets in tumor tissue samples. By taking extra caution, we carried out MSP of all the targets with the M. Sss I treated normal liver DNA as positive control templates, except for the MAGEA1 gene was unmethylated in the normal liver tissue. While only the PCR reaction designated to the unmethylated template gave rise to the detectable bands with the parental DNA, the PCR bands were evident in both reactions with the M. Sss I treated DNA (The methylation-specific PCR (MSP) is widely used for methylation profiling of the genes in human cancers for both its easiness and sensitivity. However, the necessary steps have to be taken to eliminate both false positive and negative results. Comparing the MSP-data with the non-PCR data by Southern analysis of the methylation sensitive restriction enzyme is a valuable choice, as our previous work where the hypomethylated status of both analysis . Alternaated DNA . TherefoARF, p15INK4b, p16INK4a RASSF1A, RB1 APC, BRCA1, CDH1, DAPK1, hMLH1, p14and VHL  [OCT6) [MYOD1) [RB1/p16INK4a pathway  [p27KIP1 [p57KIP2) [p53 analogue:(p73) [RASSF1A, and RASSF1C [VHL [APC [PTEN [DBCCR1) [WT1) [MAGEA1) [CAV) [CSPG2) [AR) [CDH1 [CDH13) [IRF7), and a serine/threonine kinase 1 (Peutz-Jeghers syndrome) gene (LKB1) [MGMT) [MT1A) [hMLH1 [BRCA1 [APAF1 [DAPK1 [SURVIVIN [ABL) [Eleven of the thirty four target genes were previously studied either in astrocytoma or other types of tumors. The published PCR conditions for these genes:  and VHL  were adoordingly ,24. Somely [EPO) , a ubiqu) [OCT6) , and the [MYOD1) . The majand RB1) , and two[p27KIP1  and p57Kp57KIP2) . Other gue:(p73) ,35, two  RASSF1C ), VHL [3F1C [VHL , APC [38[DBCCR1) , and the1) [WT1) . We incl[MAGEA1) , caveoli1) [CAV) , chondro [CSPG2) , androgeG2) [AR) , and cadR) [CDH1  and CDH1 [CDH13) . Three g [CDH13) , the int) [MT1A)  which pl) [hMLH1 , and BRC1 [BRCA1 , while f1 [APAF1 , DAPK1 [SURVIVIN ). FinallIN [ABL)  (AdditioABL(1), APAF1(2), APC(3), BRCA1(5), CAV(6), CDH13(8), DAPK1(11), hMLH1(14), LKB1(16), p14ARF(22), p15INK4b(23), p27KIP1(25), p57KIP2(26), PTEN (28), RASSF1C(30), RB1(31), SURVIVIN(32), and VHL(33) genes , hMLH11, LKB1163), BRCA1, CAV(6),3, BRCA1,6), CDH13, DAPK1(13) genes .Lack of hypermethylation of the RB1 gene in our observation was inconsistent with a recent report that the hypermethylated RB1 gene was detected in 19% of astrocytoma patients  . Since tPTEN gene contributed to the pathogenesis of astrocytoma [PTEN inactivation that occurs frequently in astrocytoma. This explanation might also be applicable to the no change type of methylation behavior for both the tumor associated genes (ABL(1), APAF1(2), APC(3), BRCA1(5), CAV(6), CDH13 (8), DAPK1(11), hMLH1(14), LKB1(16), p14ARF(22), p15INK4b(23), p27KIP1(25), p57KIP2(26), PTEN (28), RASSF1C(30), RB1(31), SURVIVIN(32), and VHL(33) genes) and the "cancer unrelated" genes (EPO (14)) (The genetic defects affecting the rocytoma . Lack of), hMLH11, LKB1163), BRCA1, CAV(6),3, BRCA1,), CDH13 , DAPK1(1PO (14)) .CDH1 (7), CSPG2(9), cyclin a1(10), DBCCR1(12), IRF7(15), MGMT(18), MT1A(19), MYOD1(20), OCT6(21), p16INK4a (24), p73(27), RASSF1A (39) and WT1(34)) were unmethylated in all three normal controls. In contrast, these genes were hypermethylated to various extents in the astrocytoma samples. The following six genes were marginally hypermethylated: p16INK4a, EPO, DBCCR1 and MYOD1 genes were hypermethylated in 1.9% (1/53) of astrocytoma tissues, while both CDH13 and cyclin a1 genes were hypermethylated in 5.7% (3/53) of astrocytoma cases. No significant changes of these six genes shown in here acted against the notion that DNA methylation related mechanisms underline potential inactivation of this set of genes in the pathogenesis of astrocytoma. The infrequent hypermethylation of the p16INK4a gene in astrocytoma was a total surprise, as it was frequently reported hypermethylated in various human tumors tested, including in HCC where we have previously found that the p16INK4a, MYOD1, CDH13 and cyclin a1 genes were frequently methylated [p16INK4a gene in astrocytoma was unlikely incorrect, which is consistent with a recent report that inactivation of the p16INK4a gene in 48% of astrocytoma cases was genetic [As shown in 15), MGMT, MT1A genetic .OCT6 gene was hypermethylated in 30.2% of the astrocytoma cases (16/53). Despite of the association of the OCT6 methylation with the aging process reported previously, we found no significant correlation/association of the OCT6 methylation to any clinical-pathological features, including age, gender and clinical grading of the patients. The significance of such a prevalent occurrence of the hypermethylated OCT6 gene remains to be determined. The RASSF1A  is a variant of the recently identified tumor suppressor, the RASSF1 gene that acts at downstream of the Ras mediated apoptotic pathway and is capable of binding to Ras in a GTP dependent manner [RASSF1A gene has a more extended 5' part and its promoter CpG island displays a tumor specific hypermethylated profile in a variety of tumors, HCC in particular. Furthermore, lack of the RASSF1A expression in nineteen established tumor cell lines correlates with the hypermethylated state of its promoter CpG island [RASSF1C gene has its own promoter CpG island, but is not methylated in any tumors. The methylation behavior of these two genes was very similar to our previous observation in hepatocellular carcinoma, where 22/29 cases (79%) had the fully methylated 1A along with the unmethylated 1C variants [RASSF1A promoter-CpG island was methylated in 69.8% (37/53) of astrocytoma tissues, while the C variant was not methylated in any astrocytoma tissues. The hypermethylated state of the RASSF1A promoter CpG island was not correlated with gender, age and clinical grading. Consistent with the hypermethylated status of the RASSF1A gene in U251 cells, no expression at the mRNA level was detected. Partial demethylation of its promoter by the treatment with 5-Aza-2'-deoxycytidine indeed resulted in its transcription  of astrocytoma cases. The t manner . The RASG island . The RASvariants . As show3 of astrp73 gene encodes a homologue to TP53, and loss of its heterozygosity has been observed in up to 90% of oligodendrogliomas and in 10–25% of diffuse astrocytoma [p73 gene was prevalently methylated  with no significant association with any clinical-pathological parameters, such as gender and the WHO grading. The occurrence of the hypermethylated p73 gene was more prevalent in our results than a recent report which detected the hypermethylated p73 gene in 18% (5 /28) of the WHO grade IV but not in grade III astrocytoma [p73 transcription  of cases, implying its possible involvement in the formation of astrocytoma.Both genetic defects and epigenetic abnormalities of the 's tumor . In thisIRF7) gene was analyzed. The analogue (IRF1) of IRF7 has been implicated in the IFN gamma mediated apoptosis with a profound effect on the chemo-sensitivity of tumor cells [2 = 6.632, P = 0.014). Although the gender difference remains to be understood, such a strong male association with IRF7 hypermethylation may have prognostic value.Tumor resistance to the cytotoxic chemotherapies may result from the disrupted apoptosis programs and remains a major obstacle in cancer treatment. In this study, the interferon regulatory factor 7 , and its transcription could be reactivated by demethylation with 5-Aza-2'-deoxycytidine in U251 cells  is inducible by a number of adversary agents such as heavy metals and oxidative stress. Both basal and inducible expression of this gene has been impaired in various tumor cell lines and attributed to the hypermethylated state of this gene [MT1A gene was hypermethylated in 30% (16/53) of cases, with no significant gender and grading difference. The functional and pathological implications of the MT1A hypermethylation in astrocytoma remain to be established.O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme, removes alkylating adducts from the O(6) position of guanine and protects cells from cytotoxic and mutagenic stress. Silencing of the  and p53  and is a and p53 . We founGMT gene . Expresshis gene . In thisCDH1) and H-cadherin (CDH13) has been found in parallel with the hypermethylated promoter CpG islands in various cancers [CDH1 gene was hypermethylated in 32.8% (17/53) of astrocytoma tissues, while the CDH13 gene was not methylated in all the astrocytoma tissues examined . However, its unmethylated status in all the non-astrocytoma tissues acts odd with the notion that its demethylation is associated with pathogenesis in human ovarian cancer reported previously [Although the hypermethylation mediated gene silencing of the tumor suppressor genes is at the focal point of the epigenetic studies, demethylated status of the promoter CpG islands has been linked to the tumor associated activation of the normally silenced genes -23. Ther1 in HCC  and for MAGEA1 gene [MAGEA1 gene was fully hypermethylated in all four cases of the normal liver tissues but significantly demethylated in HCC tissues . It was found fully hypermethylated in all the three control tissues and in 74.5% (40/53) of the astrocytoma tissues and partially hypermethylated  in the other astrocytoma tissues. The occurrence of the MAGEA1 demethylation in HCC differed significantly from astrocytoma . As it was fully methylated in the normal tissue, the partial hypermethylation  would imply that the event resulting in the loss of the hypermethylation state of the MAGEA1 gene indeed occurred in astrocytoma and should be scored positive for the changes in the methylation pattern in this study. The same principle has been applied for the opposite changes from the unmethylated pattern in the normal control to the partial or full hypermethylated status of all the other genes in astrocytoma tissues. It was also found that the partial demethylated status of the MAGEA1 gene in U251 cells induced by 5-Aza-2'-deoxycytidine occurred co-currently with activation of its transcription  with the clinical pathological parameters  were assessed. The methylation profiles of the AR and IRF7 genes were found gender-oriented.By statistic analysis with both Pearson Chi-Square and Fisher's Exact tests, associations of the DNA methylation profiles of the targets displaying no less than 24.5% changes . It may simply be gender associated and do not have any significant relevance to carcinogenesis of astrocytoma. It was also noticed that hypermethylation of the IRF7 gene displayed an opposite gender inclination, detected in 11% of the female patients (3/27), and 42% of male patients . Despite of the difficulty to offer a mechanistic interpretation, the potential prognostic value of such a gender-associated phenomenon might be worthwhile exploring in future.The  cancers . PhysiolThe hypermethylated status of the promoter CpG island has been linked to gene transcription silencing in a number of biological settings. The effect of the astrocytoma associated changes in the methylated state of the promoter CpG islands detected in this study on gene expression was assessed in U251 astrocytoma cells treated with the a demethylating agent, 5-Aza-2'-deoxycytidine. We used MSP to establish the methylation status of the promoter CpG island of all the genes with the astrocytoma associated methylation changes ,7,8,9 inCDH13, MAGEA1 and p73 genes were heterozygously methylated, both MGMT and RASSF1A genes were fully hypermethylated in U251 cells. The CDH13 gene was found expressed, while the rest transcriptionally inert as measured by the RT-PCR. Although both methylated and unmethylated alleles for p73 and MGMT genes were evident in U251 cells, no expression was detected, indicating that the unmethylated allele may remain silent by the other mechanisms, including the genetic defects at critical control region. By the 5-Aza-2'-deoxycytidine treatment, both demethylation of the promoter CpG island and activation of transcription of these five genes were achieved  or elevated (the CDH13 gene).As shown in panel 1 of Fig. INK4a, and RASSF1A genes, has been well established in human tumors [The DNA methylation mediated epigenetic changes also display the tumor type specific patterns, which seem to reflect the differentiation and maturation histories of the cell lineages as well as the aging process during which both global hypo- and local hyper-methylation occur. Hypermethylation of the promoter CpG islands in accord with the transcriptional silencing of the tumor suppressor genes, such as the p16n tumors . Howevern tumors , based uRASSF1A and p73 genes, hypermethylation of which was found in 20 (37.7%). Three genes that changed together were the former two plus CDH1 or OCT6, hypermethylation of which occurred in 20.8% cases (11/53)  maintained the same methylation profile as the normal control. The rest 49 cases (92.5%) suffered from the methylation changes as much as no less than one target, an occurrence was significantly lower than in HCC, where all the cases displayed methylation changes affecting no less than three targets in the studies involved with twenty or twenty four targets ,24, indiAR is associated closely with the female gender of the astrocytoma patients and devoid of any association with the formation of astrocytoma, it was taken out from this analysis. Hypermethylation of the RASSF1A gene occurred in 21 female cases . Both RASSF1A and WT1 were hypermethylated in 13 ; and the former two plus the hypermethylated p73 or CDH1 or OCT6 were found in 9 female cases , respectively ; the RASSF1A and IRF7 ; and the former two plus the p73 or MGMT or MT1A , respectively , RASSF1A plus p73 , and the former two plus either WT1 or IRF7 or MAGEA1 as well as RASSF1A plus CDH1 and WT1 . For Grade II astrocytoma, the corresponding sets consisted of the RASSF1A , the RASSF1A and MGMT or IRF7 , and the RASSF1A and MGMT plus p73 or OCT6, or MT1A, or WT1 as well as the RASSF1A and IRF7 and MT1A , respectively. For Grading III astrocytoma, those subsets were composed of the RASSF1A , the RASSF1A and CDH1 , and the formal two plus MGMT , respectively. For Grading IV astrocytoma, the comparative subsets contained the RASSF1A or p73 , the RASSF1A and p73 , and the former two plus MGMT or OCT6 , respectively. ; cyclin a1: cyclin A1; DAPK1: death-associated protein kinase 1; DBCCR1: deleted in bladder cancer chromosome region candidate 1; EPO: erythropoietin; hMLH1: mutL homolog 1, colon cancer, nonpolyposis type 2; IRF7: interferon regulatory factor 7; LKB1: serine/threonine kinase 11 (Peutz-Jeghers syndrome); MAGEA1: melanoma antigen, family A, 1 (directs expression of antigen MZ2-E); MGMT: O-6-methylguanine-DNA methyltransferase; MT1A: metallothionein 1A ; MYOD1: myogenic factor 3; OCT6: POU domain, class 3, transcription factor 1; p14ARF: the alternative reading frame of the cyclin-dependent kinase inhibitor 4a; p15INK4b: cyclin-dependent kinase inhibitor 4b; p16INK4a: cyclin-dependent kinase inhibitor 4a; p27KIP1: cyclin-dependent kinase inhibitor 1B ; p57KIP2: cyclin-dependent kinase inhibitor 1C ; p73: tumor protein p73; PTEN: phosphatase and tensin homolog; RASSF1A: ras association  domain family 1 protein isoform 1a; RASSF1C: ras association  domain family 1 protein isoform 1c; RB1: retinoblastoma 1; VHL: von Hippel-Lindau syndrome; WT1: Wilms tumor 1.HCC: Hepatocellular carcinoma; PCR: polymerase chain reaction; MSP: methylation specific PCR; None declared.JY, HYZ, JG, executing the experiments;SL and JHL, providing the patient samples;WL and YFW, carrying out the mathematic analyses of the dataJDZ: designing and organizing experiments as well as completing manuscript.The pre-publication history for this paper can be accessed here:The target promoter CpG islands and the primer pairs for methylation specific PCR. This file contains his study.Click here for filein vitro methylated genomic DNA by M. Sss I methyl transferase. The Eco RI restricted genomic DNA from the liver tissue of a healthy donor was in vitro methylated overnight with M. Sss I methyl transferase and subjected to the MSP analysis, followed by electrophoresis in a 1.3% agarose gel. *, the DNA size markers, NL, the untreated sample, U and M, MSP with the pair of primers specific to the unmethylated and methylated targets, respectively. Panels: 1, ABL; 2, APAF1; 3, APC; 4, AR; 5, BRCA1; 6, CAV; 7, CDH1; 8, CDH13; 9, CSPG2; 10, cyclin a1; 11, DAPK1; 12, DBCCR1; 13, EPO; 14, hMLH1; 15, IRF7; 16, LKB1; 17, MGMT; 18, MT1A; 19, MYOD1; 20, OCT6; 21, p14ARF; 22, p15INK4b; 23, p16INK4a; 24, p27 KIP1; 25, p57KIP2; 26, p73; 27, PTEN; 28, RASSF1A; 29, RASSF1C; 30, RB1; 31, SURVIVIN; 32, VHL and 33, WT1.Methylation profiles of thirty three genes on the Click here for fileMethylation profiles of thirty four genes in astrocytoma (part I). Both electrophoretic patterns of the representative PCR products of each of thirty four targets  and the sequencing verification of the one representative PCR product were presented. To indicate the methylation status, the sequenced data are aligned with the wild-type sequence. *, size markers, the bands of 250 bp and 100 bp were shown. U, the unmethylated; M, the hypermethylated. Panels: 1, ABL; 2, APAF1; 3, APC; 4, AR; 5, BRCA1; 6, CAV; 7, CDH1; 8, CDH13; 9, CSPG2; 10, cyclin a1; 11, DAPK1 and 12, DBCCR1.Click here for fileARF>.Methylation profiles of the promoter CpG islands of thirty four genes in astrocytoma (part II). Both electrophoretic patterns of the representative PCR products of each of thirty four targets  and the sequencing verification of the one representative PCR product were presented. To indicate the methylation status, the sequenced data are aligned with the wild-type sequence. *, size markers, the bands of 250 bp and 100 bp were shown. U, the unmethylated; M, the hypermethylated. Panels: 13, EPO; 14, hMLH1; 15, IRF7; 16, LKB1; 17, MAGEA1; 18, MGMT; 19, MT1A; 20, MYOD1; 21, OCT6 and 22, p14Click here for fileINK4b; 24, p16INK4a; 25, p27 KIP1; 26, p57KIP2; 27, p73; 28, PTEN; 29, RASSF1A; 30, RASSF1C; 31, RB1; 32, SURVIVIN; 33, VHL and 34, WT1.Methylation profiles of the promoter CpG islands of thirty four genes in astrocytoma (part III). Both electrophoretic patterns of the representative PCR products of each of thirty four targets  and the sequencing verification of the one representative PCR product were presented. To indicate the methylation status, the sequenced data are aligned with the wild-type sequence. *, size markers, the bands of 250 bp and 100 bp were shown. U, the unmethylated; M, the hypermethylated. Panels: 23, p15Click here for fileThe summary of the astrocytoma cases displaying no or changes in the methylation profiles (part I). The frequency (%) of the astrocytoma displaying no or the changes in the methylation profile of each target from the normal control were counted and presented in table as well as plotted in the figure below. The filled, shading and empty boxes indicate the cases where only hypermethylated allele, both hypermethylated and unmethylated alleles and only unmethylated alleles were detected, respectively. The frequency (%) of the hypermethylated targets (except for the MAGEA1 gene) among the total cases was scored for positive changes in astrocytoma. The MAGEA1 was fully methylated  in the control, and become partially demethylated in some astrocytoma, therefore, demethylation of the MAGEA1 in astrocytoma was scored as positive changes. Sub-tables: a, the female patient group, b, the male patient group, and c, the control.Click here for fileA1, where the heterozygously hypermethylated) among the total cases was presented in the plot.The summary of the astrocytoma cases displaying no or changes in the methylation profiles (part II). The frequency (%) of the astrocytoma displaying no or the changes in the methylation profile of each target from the normal control were counted and presented in table as well as plotted in the figure below. Sub-tables d-h, the WHO grading I to IV, respectively; The filled, shading and empty boxes indicate the cases where only hypermethylated allele, both hypermethylated and unmethylated alleles and only unmethylated alleles were detected, respectively. The frequency (%) of the hypermethylated targets .Click here for fileThe summary of changes in the methylation pattern in subsets. Both occurrence (case number) and frequency (%) for the subsets having no change in methylation and changes in one to nine genes are presented in % and (case number) in the top half of table, which was also plotted. Both occurrence (case number) and frequency (%) for the subsets having no change in methylation and changes in, at least, one to nine genes are presented in % and (case number) in the bottom half of table.Click here for fileThe summary of the concordant methylation behavior of the hypermethylated targets in astrocytoma. The co-occurrence  and frequency (%) of a panel subsets consisting of one to three targets were treated with method "Discovery Association Rules" and presented. Sub-tables: a, the total, b, the female, c, the male, and d-g, the grade I to IV, respectively. Column 1 is the number of target in each subset. Column 2 is the co-occurrence  (frequency in %). Column 3 is the occurrence of any single target in each subsets, presented in case number (frequency %). The column 4 is the gene(s) in subset. N.B., In view of the strong female inclination of the AR methylation and lacking of any association with astrocytoma, AR has been taken off from this analyses.Click here for file


We report on a woman with previously unknown left atrial myxoma, who underwent percutaneous coronary intervention. 45 months after the initial coronary angiography, echocardiography demonstrated a large atrial myxoma, which was not seen echocardiographically before. The retrospective analysis of the pre-intervention coronary angiography revealed atypical vessels in the atrial septum, which are interpreted as early signs of myxoma. Primary cardiac tumors are rare disorders, with an incidence of 0.02% at autopsy. Three quarters of the primary tumors of the heart are benign, half of which are myxomas .As noninvasive cardiac imaging becomes widely available, with increasing resolution provided by echocardiography, computed tomography and magnetic resonance imaging, cardiac tumors are being diagnosed more often. Angiography, apart from its preoperative role to rule out concomitant coronary artery disease, is rarely needed for the diagnostic work-up of cardiac tumors.This report describes the delayed presentation of a left atrial myxoma which was not depicted in an initial coronary angiography performed 51 months earlier in a woman with chest pain.A 62-year-old woman with known metabolic syndrome was referred to our clinic to exclude coronary artery disease invasively. She has been experiencing chest pain for four months which has not increased in frequency or duration since it started. She denied pain at rest, nocturnal pain, difficulty breathing, or palpitations. An echocardiographic stress examination revealed significant ischemia with anteroseptal hypokinesia. Cardiac chambers were morphologically normal. The "baseline" echocardiography and stress echo was unsuspicious of a left atrial myxoma Fig. .Coronary angiography revealed coronary artery disease with a stenosis of the proximal left anterior descending coronary artery (LAD) near the left main stem. A stent was implanted in the proximal LAD with no residual stenosis. The ventricle was morphologically normal.A coronary angiography performed 13 months after stent implantation showed no re-stenosis and a normal left ventricle. No echocardiogram was performed at this time 32 months after the control coronary angiography, the patient was readmitted because of increasing dyspnoea and palpitations. A transthoracic echocardiography disclosed a big (70 × 30 mm) mass in the left atrium attached to the interatrial septum. The tumor prolapsed into the left ventricle obstructing the mitral valve orifice  and the circumflex coronary artery (RCX). Fig. Surgery was promptly performed and the tumor was successfully excised. Histology confirmed the diagnosis of a cardiac myxoma.A retrospective analysis of the initial coronary angiographies  disclosed the atypical vessels in a small area of interatrial septum (Fig. Myxomas are benign but potentially dangerous with disturbance of rhythm, peripheral embolization or mechanical valvular obstruction, of the atrial or ventricular cavity ,3.The site, mobility, and size of the myxoma determine the clinical course. Some authors found no correlation between the size of the tumor and the clinical picture , others Myxomas presenting with systemic embolism or intracavitary obstruction can be easily detected non-invasively. The early depiction of small intracardiac tumors by means of angiography relies on the detection of atypical vessels supplied by branches of the left or right coronary artery. Our case demonstrates that this early angiographic sign is difficult to find. However, vascular malformations are not pathognomonic for myxomas  and stroIt has been reported in the literature that myxomas can be induced by radiation ,13. BetwIn the present case, a retrospective analysis of the patient angiographies disclosed the atypical vessels which were initially overseen. These vessels could have probably been interpreted as an early sign of myxoma.HPD and FK have written the manuscript and have equally contributed to this publication. HPD, VG and WR have performed the coronary angiographies. WK has performed cardiac surgery. HPD, ACB, FK and GB participated in the design and coordination of the final manuscript. All authors have read and approved the final manuscript.