SDHD that is frequently mutated in paraganglioma and pheochromocytoma, which are, like neuroblastoma, tumours originating from the neural crest. In this study, we sought for evidence for involvement of SDHD in neuroblastoma.Deletions in the long arm of chromosome 11 are observed in a subgroup of advanced stage neuroblastomas with poor outcome. The deleted region harbours the tumour suppressor gene SDHD was investigated on the genome, transcriptome and proteome level using mutation screening, methylation specific PCR, real-time quantitative PCR based homozygous deletion screening and mRNA expression profiling, immunoblotting, functional protein analysis and ultrastructural imaging of the mitochondria.SDHD mutations causing hereditary paraganglioma. No evidence for hypermethylation of the SDHD promotor region was observed, nor could we detect homozygous deletions. Interestingly, SDHD mRNA expression was significantly reduced in SDHD mutated cell lines and cell lines with 11q allelic loss as compared to both cell lines without 11q allelic loss and normal foetal neuroblast cells. However, protein analyses and assessment of mitochondrial morphology presently do not provide clues as to the possible effect of reduced SDHD expression on the neuroblastoma tumour phenotype.Analysis at the genomic level of 67 tumour samples and 37 cell lines revealed at least 2 bona-fide mutations in cell lines without allelic loss at 11q23: a 4bp-deletion causing skip of exon 3 resulting in a premature stop codon in cell line N206, and a Y93C mutation in cell line NMB located in a region affected by germline SDHD in the pathogenesis of neuroblastoma. Also, although a haplo-insufficient mechanism for SDHD involvement in advanced stage neuroblastoma could be considered, the present data do not provide consistent evidence for this hypothesis.Our study provides no indications for 2-hit involvement of  MYCN amplification and 17q gain, also have a profound predictive power [MYCN amplification and 1p-deletion in subgroup 2B and 11q-deletion often in combination with 3p-deletion in subgroup 2A [Neuroblastoma (NB) is the most frequent extra-cranial solid tumour in children, originating from immature neural crest cells of the sympathetic nervous system . The tumve power ,3. Preseve power . Subgrougroup 2A -9.SDHD, which encodes the small subunit D (cybS, cytochrome b558) of the mitochondrial respiratory chain complex II (succinate-ubiquinone oxidoreductase) [The first evidence for the occurrence of 11q-deletions in NB was obtained in 1991 . Howeverductase) ,18 and wSDHD in tumour development was obtained by the discovery of germline mutations in this gene as the cause for familial paraganglioma (PGL) [SDHD mutations were also detected in patients with apparently sporadic pheochromocytoma (PC) [SDHD mutations in the 5' portion of the gene causing complete disassembly of complex II, whereas PGL are associated with mutations in the 3' region of the gene causing partial inactivation of its catalytic activity [SDHD in oxidative phosphorylation, SDHD has also been presumed to contribute to the function of the mitochondria as oxygen sensors. It was shown that SDHD inactivation leads to a pseudo-hypoxic state and upregulation of hypoxia responsive genes, possibly through increased production of reactive oxygen species (ROS) [SDHD mutations or reduced activity of SDHD might lead to impaired oxidative phosphorylation and hypoxia and thus contribute to NB oncogenesis.The first evidence for a role of ma (PGL) . Somaticoma (PC) ,21. It sactivity -28. PGL es (ROS) . A hypoxes (ROS) . ConsequSDHD as a positional and functional candidate for the presumed NB tumour suppressor gene on 11q23. In order to search for evidence for involvement of SDHD in NB development, an extensive series of investigations was performed on the DNA, RNA and protein level.In view of the above, we considered MYCN amplification. For all NB patients constitutional leukocyte DNA was available. In addition, 31 NB cell lines were included in the analysis of which karyotypes were available. For 20 of these cell lines, comparative genomic hybridization (CGH) data and/or M-FISH (multicolour fluorescence in situ hybridization) results have been published [Neuroblastoma (NB) tumour samples (at least 70% tumour cells) were collected at the Ghent University Hospital (Ghent, Belgium) n = 32) and in the Molecular Oncology Unit (Lyon, France) (n = 35). Ethical approval was obtained for the collection of the tumour samples. The latter group includes selected patients with stage 3 or 4 NB without ublished -32. For  and in tublished .SDHD transcripts. RNA of the cell line pellets (treated and untreated) was extracted with the RNeasy Mini kit (Qiagen) according to the manufacturer, followed by RNase free DNase treatment on column (Qiagen). A fraction of the untreated NB cell line cultures was also used for functional enzyme assays.Cultures of NB cell lines N206, SK-N-AS, SK-N-SH, NMB, SK-N-FI, CLB-GA, LA-N-2 and NGP were treated with puromycine (100 μg/ml) during 6 hours in order to prevent possible nonsense mediated RNA decay of variant MLL (11q23.3) SpectrumOrange probe (Vysis) and BAC clone RP11-93E4 for the CRTAM gene (11q24.1) in combination with a centromeric probe for chromosome 11. Labelling and FISH was performed as described [The 11q status of the cell lines was evaluated using FISH. FISH was performed using the LSI escribed . For eacSDHD gene. In order to discriminate between whole chromosome loss, and unbalanced 11q loss , two microsatellite markers on 11p (D11S922 on 11p15.5 and D11S1324 on 11p14.1) were analyzed in patients that showed allelic imbalance for all 11q markers (positions of the markers are according to the UCSC Genome Browser, freeze version July 2003). Scoring of loss of heterozygosity (LOH) was performed by calculation of the allelic imbalance factor (AIF) [All NB patients were analyzed with 4 microsatellite markers on 11q23: D11S1986 (11q23.1), D11S1998 (11q23.3), D11S1356 (11q23.3) and D11S1299 (11q23.3), of which D11S1986 and D11S1998 are immediately flanking the SDHD using Primer Express v2.0 (Applied Biosystems)  Table . Exon 1 [ . Primers were designed in a CpG island close to the start of the gene (putative SDHD promotor) (chr11: 111495002–111495330: UCSC Genome Browser freeze version July 2003) (methylated forward 5'GTAGTCGGGATCGAGTATTAGTGAGTC3', methylated reverse 5'AATAAACCGAAAATCGAAAAACGAT3', unmethylated forward 5'AGTTGGGATTGAGTATTAGTGAGTTGT3', unmethylated reverse 5'ACTAAATAAACCAAAAATCAAAAAACAAT3'). Amplification mixtures (50 μl) for the PCR reaction contained 50 ng template DNA, 1× Platinum Taq PCR reaction buffer (Invitrogen), 6 mM MgCl2, 200 μM of each dNTP, 1.25 U Platinum Taq polymerase (Invitrogen), 3% DMSO and 300 nM of each primer. The cycling conditions comprised 4 min polymerase activation at 93°C, 40 cycles with denaturation at 93°C for 30 sec, annealing at 64°C (methylated primers) or 65°C (unmethylated primers) for 30 sec and extension at 72°C for 30 sec, and a final extension for 5 min at 72°C. SssI methylase (New England Biolabs) treated DNA, following the manufacturer's instructions and normal human genomic DNA were used as a positive and negative control respectively after bisulfite modification.On the 31 NB cell lines and on another series of 50 NB tumours of which 15 were included in the mutation analysis, methylation-specific PCR (MSP) was performed as described, with minor modifications . MSP pri and checSDHD exons were designed using Primer Express v2.0 (Applied Biosystems), based on the publicly available SDHD genomic sequence (accession number AB026906) (Table 2, 200 μM of each dNTP, 1 U Platinum Taq polymerase (Invitrogen) and 500 nM of each primer. The cycling conditions comprised 3 min polymerase activation at 94°C, 35 cycles with denaturation at 92°C for 20 sec, annealing at 60°C for 20 sec and extension at 72°C for 2 min, a final extension for 5 min at 72°C and a slow decrease in temperature to 25°C over 30 minutes. One μl of the PCR products was analyzed on a Ready-To-Run Agarose Gel (1.2%) (Amersham Biosciences).Intronic primers flanking the m(= 62.1°C)+0.7°C and Tm+1.5°C. Exon 2 fragments were eluted at a temperature of Tm(= 55.7°C)+4.8°C. Exon 3 fragments were eluted at a temperature of Tm(= 57.4°C)-0.4°C, Tm+1.1°C and Tm+3.8°C. Exon 4 fragments were eluted at a temperature of Tm(= 56.4°C)-0.4°C, Tm+1.6°C and Tm+3.2°C. Elution of the fragments was performed using standard conditions according to the manufacturer. Elution profiles were analyzed using the Wavemaker software.Denaturing high-pressure liquid chromatography (DHPLC) was performed using the Wave system (Transgenomic). The melting profile of each fragment was determined using the Wavemaker software v4.1 (Transgenomic). Crude PCR product was injected into a preheated, fully equilibrated chromatographic column for the DHPLC analysis. Exon 1 fragments were eluted at a temperature of TSequencing was performed on all cell lines and on tumour samples with aberrant DHPLC elution peaks .Amplified fragments were purified using the Montage PCR96 filter plates (Millipore) or by excision of the fragment of interest from a 1.5% TBE-agarose gel and purification on a GenElute Minus EtBr Spin Column (Sigma-Aldrich). Cycle sequencing was performed using purified amplicons (3–10 ng), the above-mentioned primers Table  at a conPCR primers and minor groove binder (MGB) probes for sequence variant IVS4-32T>C were designed using Primer Express v2.0 following the user bulletin guidelines for the design of MGB probes (Applied Biosystems): forward primer 5'TTTTTTGCAGCCAAGTTATCTGTATAG3', reverse primer 5'TGTCCAAGGCCCCTAAAGAA3', MGB probe allele 1 5'TGTGGTTTTTtATTGATG3' labelled with 6-FAM and MGB probe allele 2 5'TGTGGTTTTTcATTGAT3' labelled with VIC. To address the frequency of the sequence variant g.7876A>G (Y93C) in a normal population, the following primers and probe were designed: forward primer 5'GGCTGCTTATTTGAATCCTTGCT3', reverse primer 5'ACTTGCCAGTGACCATGAAGAGT3' and MGB probe variant allele 5'ATGGACTgTTCCCTG3' labelled with VIC. The reaction mixture contained 10 ng of DNA, 100 nM of each MGB probe, 300 nM of each primer and 1× qPCR Mastermix (Eurogentec). For the screening of the g.7876A>G variant, multiplex PCR was performed using primers and probe of the normal allele of the above-mentioned SNP (IVS4-32T>C) and primers and probe for the variant allele g.7876A>G. Reactions were performed on the iCycler Thermal Cycler (Bio-Rad) with the following thermocycling conditions: an initial activation step at 95°C for 10 min, 50 cycles of 95°C for 15 sec and 60°C for 1 min. Allelic discrimination data analysis was performed on the iCycler IQ Optical System Software v3.0a (Bio-Rad).SDHD_IVS4-32).The SNP info for the IVS4-32T>C variant was submitted to NCBI's SNP database (sn#5606973, SDHD sequence variants present in other cell lines, the coding region of the full-length SDHD mRNA was amplified for cell lines N206, SK-N-AS, SK-N-SH, NMB, SK-N-FI, CLB-GA, LA-N-2 and NGP, before and after puromycin treatment. RNA extraction, DNase treatment and cDNA synthesis were performed as described [SDHD.In order to investigate predicted or putative splice variants caused by the 4 bp-deletion in NB cell line N206 and escribed . SubsequSDHD expression levels were determined using an optimized two-step SYBR Green I RT-PCR assay [T method was used for quantification. PCR reagents were obtained from Eurogentec as SYBR Green I mastermixes and used according to the manufacturer's instructions. Primers in exon 3 were designed using Primer Express (see Primers Exon 3 in Table UBC, HPRT1, SDHA and GAPD) as reported previously [Relative CR assay  with minCR assay . The comeviously .Enzyme activities were determined spectrophotometrically as previously described .Protein amount was determined with immunoblot analysis of complex II Fp fragment as previously described . Relativ4 for 1 h at 4°C. Block staining in uranylacetate (UAc) in 70 % ethanol was followed by dehydration in ethanol and propylene oxide, and embedding in Epon. Ultrathin sections were counterstained with UAc and lead. At least 2 cells in each culture were photographed at a magnification of 20,000 in a Zeiss electron microscope operating at 50 KV. Per culture, 25–63 mitochondria were examined. Their projected length (largest straight distance) was measured and matrix electron density was compared to the surrounding cytosol.Mitochondria of NB cell lines LA-N-2, SK-N-AS, CLB-GA, NGP, CHP-901, SK-N-SH, SK-N-FI, N206, SJNB-12, SJNB-8, NMB, SJNB-10 and IMR-32, breast carcinoma cell line MCF-7 and, Ewing sarcoma cell line SK-N-MC were analysed by electron microscopy. Monolayers from these cell lines were briefly rinsed twice in PBS and then immersed at room temperature in 3 % glutaraldehyde buffered with Na-cacodylate at pH 7.3 for 1 h. After rinses in this buffer with 1 % bovine serum albumin, cells were scraped off with a rubber policeman, and centrifuged with 3 % glutaraldehyde. After washing, the pellets were postfixed in 2 % buffered OsOElectron microscopic files were made on 11 archived neuroblastic tumours. In addition, previously published cases were examined for mitochondria morphology.MLL locus (11q23.23) (not shown) . No previously unnoticed submicroscopic 11q23-deletions were detected. Screening for homozygous deletions in all SDHD exons was negative for the 31 NB cell lines.Previous karyotyping, comparative genomic hybridization (CGH) and/or M-FISH revealed 11q-deletions in 9 out of 31 neuroblastoma (NB) cell lines (CLB-GA, GI-ME-N, IMR-32, LA-N-6, NBL-S, NGP, SK-N-AS, NMB, N206) -32. In tMYCN amplification, in contrast to the other patient subgroup for which samples were unselected.In 20 of the 67 NB tumour samples, loss of heterozygosity (LOH) or allelic imbalance (AI) (AIF > 2) in the 11q23 region was found  analysis and subsequent sequencing of the Two variants were considered as bona fide mutations Figure . The firIn one patient without 11q allelic loss (F11) we observed in both tumour and constitutional DNA a TCTA insertion at position IVS2+37. However, no additional tumour material nor parental material was available for further analysis. So, it remains unclear whether this is a true mutation or a rare polymorphism.In addition, 1 new and 4 known polymorphisms were observed. The H50R variant found in cell line LA-N-2 was described as a polymorphism in several studies -45. ThisSDHD alleles has been deleted, indicates that all three variants are located on the same allele, representing a low frequent haplotype.The presence of the IVS3-29A>G, S68S and IVS4-32T>C variants in a cell line (NGP) and two tumours (F18 and F35), in which one of both SDHD promotor hypermethylation was tested for 31 NB cell lines and 50 NB patients using methylation-specific PCR (MSP). No evidence for methylation was obtained in any of the analyzed NB cases.SDHD cDNA showed that a 4 bp deletion in the intron-exon boundary in cell line N206 caused skipping of exon 3 leading to a premature stop codon.Amplification of the full-length No alternative transcripts could be detected in cell lines NMB, SK-N-FI, NGP and LA-N-2 carrying basepair variants (and 3 control cell lines without sequence variants SK-N-SH, SK-N-AS and CLB-GA) when grown with or without puromycin Figure .SDHD is bi-allelically expressed, thus supporting recent observations in lymphoblastoid cell lines, adult kidney and adult and fetal brain [The above-mentioned cDNA transcript sequencing revealed that al brain ,22, but SDHD expression levels were measured using real-time quantitative PCR in 31 NB cell lines, normal foetal neuroblast cells  and 7 normal adult tissues (brain, heart, kidney, liver, lung, trachea and breast)  and normal adult tissue mRNA samples (Mann-Whitney test: P = 1.49E-05).) Figure . The SDHSDHD mRNA levels were significantly reduced in cell lines with 11q allelic loss and SDHD mutated cell lines (i.e. NMB and N206) (N = 9) compared to cell lines without 11q allelic loss (N = 22) (Mann-Whitney test: P = 1.49E-03).SDHD gene encodes the small subunit D of the mitochondrial respiratory chain complex II we decided to assess the effect of the basepair variants on the activity of complex II of the respiratory chain by spectrophotometrical measurements in 5 NB cell lines (N206, NMB, SK-N-FI, NGP and LA-N-2) and 3 control NB cell lines without sequence variants (SK-N-SH, SK-N-AS and CLB-GA). No significant differences in complex II enzyme activity could be demonstrated. Although, in LA-N-2 a slight decrease of complex II activity was observed (data not shown).As the On above-mentioned cell lines and NB cell lines CHP-901, SJNB-12, SJNB-8, SJNB-10 and IMR-32, breast cancer cell line MCF7 and Ewing sarcoma cell line SK-N-MC, immunoblotting of the Fp fragment of complex II showed no significant variation in abundance among the tumour cell lines (data not shown).Electron microscopic analysis of NB cell lines revealed that the morphology of the mitochondria is heterogeneous between the different cell lines, with respect to length, dilated intracrista spaces and condensation of the matrix Table . In mostIn order to examine whether dilated mitochondrial cristae are a feature of many, or all NBs, we studied the mitochondria in electron micrographs from 11 archived and 22 previously published neuroblastic tumours -53. DilaSDHD in neuroblastoma (NB) tumourigenesis. In a first step, mutation and methylation analyses were performed on a large panel of NB cell lines and tumours. A total of seven sequence variants (in nine different samples) were detected of which two could represent bona fide mutations, i.e. missense mutation Y93C in cell line NMB and a 4 bp deletion in cell line N206.In this study, we investigated the possible involvement of SDHD protein frequently altered due to germline mutations in paraganglioma (PGL) families  and consequently the structure of the transmembrane subunit and/or association of the catalytic domain subunits SDHA and SDHB to the membrane would be disrupted .The functional consequence of one sequence variant located within an intronic sequence (IVS2+37ins(TCTA)) is more difficult to evaluate due to lack of fresh tumour material, and parental DNA. Further analysis is needed in order to reveal a possible effect on splicing or RNA stability.Finally, one new and 4 known polymorphisms were detected in 5 NB cell lines and 3 tumour samples. Additional screening for homozygous deletions in all cell lines and methylation in cell lines and tumours were negative.SDHD as a classical tumour suppressor gene in NB. However, the finding of two apparently bona fide SDHD mutations in NB without allelic loss of distal 11q leaves the possibility open that the gene contributes to NB oncogenesis due to haplo-insufficiency, rather than functional inactivation of both alleles. In order to investigate this possibility, we decided to perform further studies at transcript and protein level. Interestingly, SDHD expression was shown to be consistently lower in cell lines with 11q allelic loss versus NB cell lines without loss and also significantly decreased in NB cell lines as compared to normal foetal adrenal neuroblast cells of 16, 18 and 19 weeks gestational time with a mean fold difference of 3.61 between neuroblast cells and NB cell lines. A similar correlation between 11q LOH and reduced SDHD expression was recently described in colorectal and gastric cancer [SDHD mutation. However, measurement of complex II activity might only reflect part of the functional properties of SDHD. Also, measurement of differences in protein quantity is far less sensitive than Q-PCR at transcript levels. Therefore, these observations at present do not fully exclude SDHD involvement in NB. Finally, we also looked at the morphologic characteristics of the mitochondria as a possible clue to SDHD dysfunction. In keeping with, at best, partial loss of function of SDHD, we did not observe similar gross morphologic changes as reported for PGL with SDHD mutations , the latter being characterized by destabilization of complex II with loss of enzymatic activity [Based upon these results, we can exclude a role for c cancer . Our finactivity . Howeveractivity . Subsequactivity ,58. ThisSDHD involvement in NB. However, the finding of, albeit rare, bona fide mutations and reduced expression of SDHD in NB with 11q allelic loss hints at a possible haplo-insufficient contribution to tumour development. A better understanding of the different functions of SDHD, in particular its possible contribution to energy independent apoptosis involving the release of cytochrome c and procaspases, will allow further functional assays to asses how this gene contributes to tumour development in general, and the high stage NB phenotype in particular [In contrast to previous findings in PGL and PC, this study excludes a classical two hit Knudson model for rticular ,60.CDKN1B (p27Kip1) [TP53 (p53) [DMP1 [PTEN [APC [NKX3.1 [SDHD in NB, a bipartite mechanism as tumour suppressor gene for the SDHD gene, as described for the APC gene can at present not be fully excluded. Following this hypothesis, germline mutations in SDHD would predispose to PGL or PC development. Rare somatic mutations and more typically loss of one allele could contribute to the metastasizing NB tumour phenotype , not as an initiating step but rather as later event in tumour development. However, further evidence is needed to support the haplo-insufficient involvement of SDHD in cancer. Ultimately, knockout mice for the SDHD gene leading to haplo-insufficiency for SDHD in neuroblast progenitor cells, would be the appropriate test to evaluate this hypothesis.Evidence for contribution to a cancer phenotype through haplo-insufficiency has recently been obtained for a number of loci, including p27Kip1) ,62, TP5353 (p53) , DMP1 [63) [DMP1 , PTEN [6P1 [PTEN , APC [66 [NKX3.1 . In mous [NKX3.1 . AlthougAIF = allelic imbalance factorCGH = comparative genomic hybridizationDHPLC = denaturing high performance liquid chromatographyLOH = loss of heterozygosityMGB = minor groove binderMSP = methylation specific PCRNB = neuroblastomaPC = pheochromocytomaPGL = paragangliomaROS = reactive oxygen speciesSDHD = succinate dehydrogenase, subunit DSNP = single nucleotide polymorphismSRO = shortest region of overlapThe authors declare that they have no competing interests.KDP carried out the genomic and transcriptomic studies, and drafted the manuscript. JH performed the methylation studies. JS carried out the immunoblottings and spectrophotometric analysis that was evaluated by RVC. AN performed the ultrastructural analysis that was screened and discussed by CV, FR and MP. GL and NVR collected the tumour material. JV and FS participated in the study's design and coordination. All authors have reviewed the manuscript and FS and ADP were the final editors of the manuscript.The pre-publication history for this paper can be accessed here:


The problems associated with the ageing immune system coupled with possible solutions were discussed recently at the British Society for Immunology Annual Congress in Harrogate in December 2004. The session "Ageing and the Immune System in vivo" dealt in details with the immune risk phenotype and the potential methods of reversing the problems of an ageing immune system. This is a commentary on that session. From an early age our immune system is introduced to a variety of infectious agents through contact with infected individuals. We also deliberately present our immune responses either to attenuated organisms or to components of infectious pathogens in order to provoke a response. A successful response to these organisms when first encountered provides us with immunological memory to enable our immune systems to make more rapidly responses to the same potentially infectious agents at a later date. In theory the longer we live the better our memory responses and our ability to cope with any potential pathogen seen previously. Since there are no completely sterile environments, individuals who have survived to reach a ripe old age must have combated many possible infectious agents, and have an immune system with a prodigious memory component. Only part of this is true. As we age the memory component of our immune system, as measured by the number of memory cells, does increase. Unfortunately this is not accompanied by improved immunity even to infectious agents that have been overcome earlier in life -4.The problems associated with the ageing immune system coupled with possible solutions were discussed recently at the British Society for Immunology Annual Congress in Harrogate in December 2004. The session "Ageing and the Immune System in vivo" dealt in details with the immune risk phenotype and the potential methods of reversing the problems of an ageing immune system.+CD28- cells (or CD8+CD28-CD27- in the very old), low numbers of B cells and the presence of CD8 T cells which were cytomegalovirus (CMV) tetramer positive. Within both the OCTO and NONA groups Dr Anders was able to show that the immune risk phenotype had some predictive input towards morbidity. This was even more apparent when cognitive impairment was included in the calculations.The concept of the immune risk phenotype was described by Dr Anders Wikby (Jönköping University) who carried out a longitudinal immunological study in order to establish predictive factors for longevity. Dr Anders first worked on a group of normal octogenarians in the OCTA study in Sweden which later became the NONA study as these individuals entered their nineties. Within this group he was able to describe the immune risk phenotype which he characterised by a CD4:CD8 ratio of <1, poor in vitro T cell proliferation, an increase in the number of CD8+ and show an increased incidence with age, so far it seems that clonal expansion is not due to malignant transformation but may follow antigen stimulation. The control of CMV within the body is mainly attributed to CD8+ T cells and Professor Moss revealed that in older individuals up to 50% of CD8+ T cells may be specific for CMV as judged by tetramer staining. It is possible that this increased response to CMV may lead to the impairment of responses to other viruses in the aged. This theme of CMV infection and ageing was then continued by Professor Graham Pawelec (University of Tuebingen) who introduced the use of the KLRG-1 marker as a marker of senescence. T cells which are KLRG-1+ do not proliferate in vitro when provided with a stimulus which would induced proliferation in T cells which lack this marker. In elderly individuals fewer CMV tetramer positive CD8+ T cells secreted interferon-γ in Elispot assays when compared with similar cells from younger individuals. Furthermore between 96 and 99%of the CD8+ T cells which stained with the CMV tetramer also expressed the KLRG-1 marker. The theme of CMV infection in the elderly was further continued by Professor Arne Akbar (University College London) whose research group has recently been analyzing telomere lengths in different T cell subsets using a fluorescent staining technique. In this method telomeres are stained with a fluorescently labeled probe and the brighter the fluorescent labeling the longer the telomere. Using this technique Professor Akbar reported that CMV specific CD4+ T cells in older individuals had very short telomeres indicating their limited replicative capacity and that more of these CMV specific CD4+ T cells expressed interferon-γ.The effect of CMV infection on the immune system in the elderly was also discussed by Professor Paul Moss (University of Birmingham) with particular emphasis on the clonal expansion of T cells. Clonally expanded T cells are usually CD8This view of a bleak future for displaying the immune risk phenotype was countered by the last two presentations which discussed different approaches to reversing the defects seen in the immune system with age. Both of these methods centered around reversing the atrophy of the thymus seen with age. Dr Jayne Sutherland, currently at the Anthony Nolan Research Institute, detailed her work carried out whilst with Professor Richard Boyd (Monash University) on reversing age associated thymic atrophy by surgical or chemical castration. Studies in mice revealed that reversal of thymic atrophy followed surgical castration and in humans chemical castration produced changes in the TREC levels which indicated improved thymic output. Functional studies showed that this treatment improved immune function in the treated aged subjects. This theme of improved immunity following reversal of thymic atrophy was carried further in work reported by Dr Richard Aspinall . He showed how therapeutic intervention with interleukin 7 and derivates could reverse atrophy of the thymus in old animals and also lead to improved immune function compared with age and sex matched controls. In addition he described work done in the Gambia which suggested a link between the development and enlargement of the thymus after birth and the level of interleukin 7 in breast milk.Two speakers from the posters presentations were also chosen to present their results. K. Wang from Professor Janet Lord's group (Birmingham) presented work on dehydroepiandrosterone, (DHEA). This steroid hormone declines with age and treatment of NK cells with DHEA increases NK cytotoxicity and further analysis shows that DHEA induces PKC-β translocation and upregulates perforin expression in these cells. D. Silva from Donald Palmers laboratory  presented work on the expression of neuropeptides in epithelial cells from the thymuses of different species.The meeting revealed that much needs to be done in characterizing the changes in the immune system which are associated with ageing, and members of the audience were asked whether they were interested in receiving more details about the newly formed "Differentiation and Immunosenesence Affinity Group" associated with the BSI.


One of the central problems for much of the 20th century was how to reconcile genetic stability with evolutionary change. Genomic fidelity was thought to arise from an inherent invariability in the DNA structure itself. Biologists now know that DNA constantly undergoes modifications as it unwinds, replicates, condenses, twists, and untwists. This dynamic interplay produces both stability and variation—and occasionally genetic damage. If DNA damage goes unrepaired, it can disrupt chromosomal integrity and may lead to cancer and other diseases. When the DNA double helix breaks, the cell must enlist a number of proteins to repair the broken DNA ends, but much remains to be learned about the molecular mechanisms involved. Tracking a protein that binds to single strands of DNA during replication and recombination in living yeast cells, Xuan Wang and James Haber report that this protein plays a role in at least two key steps in the repair of double-strand breaks in DNA.When double-strand breaks occur, the cell mounts a search for similar (homologous) sequences that can be used as a template to repair the damaged sequence. If successful, the broken DNA molecule basepairs with the homologous region and forms a complex, ultimately replacing the damaged sequence with a similar sequence. In yeast—which serves as a stand-in for higher eukaryotes, including humans—this “strand invasion” process requires both an exchange protein, called Rad51, and a single-stranded DNA-binding protein, called RPA (replication protein A). Single-stranded binding proteins bind to regions of DNA that are opened up during replication. They also bind to strands when broken ends of DNA are cut by enzymes that leave long single-stranded tails. RPA proteins are thought to facilitate the formation of Rad51 polymers, or filaments, on single-stranded DNA by clearing away structures that block Rad51's path. The growing filament searches for homologous DNA sequences and promotes the invasion of the single strand, preparing it to copy the homologous template by “repair DNA synthesis,” which patches up the lesion.To investigate how RPA functions in double-strand break repair in a living organism, Wang and Haber created cells with a double-strand break at a specific site and monitored the activity of proteins recruited to repair the damage. With this approach, the researchers could observe these interactions in living yeast to determine what role RPA plays in repairing DNA damage and how it works with the Rad51 protein.The authors show that as soon as a double-strand break occurs, the RPA protein binds to the exposed strand ends, before the Rad51 protein does. This is not unexpected, because this binding order supports the model that RPA prepares the way for Rad51, perhaps by stabilizing the strand long enough for Rad51 filaments to establish themselves. The surprise was that RPA appears to be necessary even after Rad51 binds to the DNA strand, perhaps by stabilizing the interaction with homologous DNA sequences. That RPA is required for successful repair is supported by evidence that a particular mutated form of RPA can stimulate Rad51 DNA binding normally, but inhibits strand exchange and template copying, thus preventing repair of DNA damage.Wang and Haber's work highlights the complex repertoire of DNA–protein and protein–protein interactions that manage and manipulate the genome in the service of genomic stability. The study of DNA repair mechanisms in living cells—a daunting task—promises to lend valuable insights into the truly dynamic nature of maintaining genome stability.


In situ apoM mRNA hybridization demonstrates that apoM is exclusively expressed in the hepatocytes and in the tubule epithelial cells in kidney. Expression of apoM could be regulated by platelet activating factor (PAF), transforming growth factors (TGF), insulin-like growth factor (IGF) and leptin in vivo and/or in vitro. It has been demonstrated that apoM expression is dramatically decreased in apoA-I deficient mouse. Hepatocyte nuclear factor-1α (HNF-1α) is an activator of apoM gene promoter. Deficiency of HNF-1α mouse shows lack of apoM expression. Mutations in HNF-1α (MODY3) have reduced serum apoM levels. Expression of apoM is significantly decreased in leptin deficient (ob/ob) mouse or leptin receptor deficient (db/db) mouse. ApoM concentration in plasma is positively correlated to leptin level in obese subjects. These may suggest that apoM is related to the initiation and progression of MODY3 and/or obesity.Apolipoprotein M (apoM) is a 26-kDa protein that is mainly associated with high-density lipoprotein (HDL) in human plasma, with a small proportion present in triglyceride-rich lipoproteins (TGRLP) and low-density lipoproteins (LDL). Human apoM gene is located in p21.31 on chromosome 6 (chromosome 17, in mouse). Human apoM cDNA (734 base pairs) encodes 188-amino acid residue-long protein. It belongs to lipocalin protein superfamily. Human tissue expression array study indicates that apoM is only expressed in liver and in kidney and small amounts are found in fetal liver and kidney.  Human apolipoprotein M (apoM) was found and initially isolated from chylomicrons by Xu and Dahlbäck in 1999 . Gel filThe identified human apoM cDNA (734 base pairs) encoded 188-amino acid residue-long protein. The 5'-untranslated region was 33 nucleotides and the 3'-untranslated region 120 nucleotides, not including the poly (A) tail. Southern blot analysis of different species gave positive signals in all mammalian genomes but not in DNA from chicken and yeast [Based on sensitive sequence searches, it is proposed that apoM is related to the lipocalin protein superfamily Fig.  1]. Sub. Sub1]. in situ hybridization. In day-15 embryos, apoM was expressed in both liver and kidney. During human embryogenesis, apoM was strongly expressed in livers of 3–5 month-old human embryos and continued to be strongly expressed throughout embryogenesis. In the kidney, apoM expression was highest in 5–9 month-old embryos. There was some expression of apoM in small intestine, particularly in later stages of embryogenesis. In skeletal muscle, minute apoM expression was found in 3–5 months-old embryos, and some apoM expression was found in stomach in earlier stages of embryogenesis [in situ apoM mRNA hybridization demonstrated that apoM is exclusively expressed in the hepatocytes and in the tubule epithelial cells in human kidney [in vivo, which may be related to the hepatic lipid and/or lipoprotein metabolism.Northern blot analyses of multiple tissues  showed that apoM was mainly expressed in kidney and liver . Furtherogenesis . These fIn vitro, several biological factors have been tested to examine their influences over the transcription and secretion of apoM in hepatic cell line (HepG2 cells). Like apoB, apoM is highly hydrophobic and must co-circulate with lipoprotein particles in the blood stream. It has been demonstrated that apoB could be down-regulated by transforming growth factor-beta (TGF-β) [in vivo. In another study, Xu et al demonstrated that platelet-activating factor (PAF) could up-regulate apoM expression in HepG2 cells, whereas, lexipafant, a PAF-receptor antagonist significantly suppressed the mRNA levels and the secretion of apoM in HepG2 cells in a dose-dependent manner. Neither tumor necrosis factor-α (TNF-α) nor interleukin-1α (IL-1α) influences apoM expression or secretion in HepG2 cell cultures [ (TGF-β) ,8. Xu et (TGF-β) . It suggcultures . It indiin vivo and in vitro [in vivo. Richter et al. reported that apoM gene expression could be regulated by HNF-1α. Mutant HNF-1α-/- mice completely lack expression of apoM in liver and kidney. Serum apoM levels in HNF-1α+/- mice are reduced by 50% compared with wild-type animals. By analyzing the apoM promoter and identifying a conserved HNF-1 binding site, they showed that HNF-1α is a potent activator of apoM promoter, that a specific mutation in the HNF-1 binding site abolished transcriptional activation of apoM gene. HNF-1α protein can bind to the HNF-1 binding site of apoM promoter in vitro [ob/ob mice [ob/ob mice and in leptin-receptor deficient db/db mice than in control mice. Furthermore, leptin administration significantly increased plasma apoM levels and apoM mRNA levels in liver and in kidney in ob/ob mice [in vivo.Administration of adrenocorticotropic hormone (ACTH) has beneficial effects on plasma lipoproteins -15. A coin vitro ,19, indiin vitro . Liang a/ob mice , suggest/ob mice . Xu et a/ob mice . It is c2, n = 51). In univariate analysis, apoM correlated significantly to leptin (r = 0.54, P < 0.001), BMI (r = 0.70, P < 0.001), fasting insulin (r = 0.33, P = 0.025), total cholesterol (r = -0.41, P = 0.016), and LDL-cholesterol (r = -0.39, P = 0.018). The correlations between apoM and cholesterol and between apoM and leptin remained significant after adjustment for the influence of BMI. Forward stepwise multiple regressions when leptin, BMI, insulin and cholesterol were entered in a model as independent variables and apoM as the dependent variable showed that cholesterol and leptin were independent predictors of circulating apoM. These two parameters yielded an r2 of 0.28, thereby explaining approximately 30% of the variance in apoM. Hence, apoM is positively correlated to leptin and negatively correlated to cholesterol levels in humans [Xu et al. investigated the relationship between plasma apoM levels and leptin levels, body mass index (BMI), fasting glucose, fasting insulin as well as lipoprotein concentrations in females displaying a wide range in BMI (18.9–57.1 kg/mn humans . Richtern humans . Alzheimn humans -26. Incrn humans ,28, suggn humans . With thin vivo and in vitro. HNF-1α is one of the most important activator of apoM gene promoter. Plasma apoM concentration is positively correlated to leptin levels and negatively related to plasma cholesterol levels. Both leptin and leptin receptor are essential for apoM expression in vivo. Plasma apoM levels may be used as the marker for identification of MODY3. The detailed relationship between apoM and MODY3 as well as obese needs further investigation.In conclusion, apoM is a novel HDL apolipoprotein. Like apoB apoM could be regulated by several cytokines


Bacillus subtilis MreB and Mbl have been shown to perform dynamic motor like movements within cells, extending along helical tracks in a time scale of few seconds.Bacterial actin-like proteins have been shown to perform essential functions in several aspects of cellular physiology. They affect cell growth, cell shape, chromosome segregation and polar localization of proteins, and localize as helical filaments underneath the cell membrane. Bacillus subtilis MreB has a dual role, both in the formation of rod cell shape, and in chromosome segregation, however, its function in cell shape is distinct from that of MreC. Additionally, MreB is important for the localization of the replication machinery to the cell centre, which becomes aberrant soon after depletion of MreB. 3D image reconstructions suggest that frequently, MreB filaments consist of several discontinuous helical filaments with varying length. The localization of MreB was abnormal in cells with decondensed chromosomes, as well as during depletion of Mbl, MreBH and of the MreC/MreD proteins, which we show localize to the cell membrane. Thus, proper positioning of MreB filaments depends on and is affected by a variety of factors in the cell.In this work, we show that Our data provide genetic and cytological links between MreB and the membrane, as well as with other actin like proteins, and further supports the connection of MreB with the chromosome. The functional dependence on MreB of the localization of the replication machinery suggests that the replisome is not anchored at the cell centre, but is positioned in a dynamic manner. In vitro, actin filaments can deform vesicles and thus push membranes, providing the force to elongate cellular extensions such as pseudopods [Listeria monocytogenes cells move within macrophages through propelling by actin bundles that extend at only one pole of the bacterial cells, due to the ActA protein that is present at one cell pole and that induces rapid polymerisation of actin. Bacterial cells also possess several different actin-like proteins [Actin provides vital functions as a cytoskeletal component in eukaryotic and in prokaryotic cells. In eukaryotes, actin filaments give mechanical strength to cells in form of a dynamic cytoskeleton, and are structural fibers in muscle contraction. Additionally, actin proteins have motor like functions -3, most eudopods ,5. Listeproteins . MreB isproteins , the truBacillus subtilis MreB or of Mbl, the second actin ortholog, or of Caulobacter crescentus MreB, origin regions on the chromosomes fail to separate properly, leading to a severe (or in case of Mbl more moderate) segregation defect [mreB allele in E. coli [E. coli ParR protein binds to a specific cis site on the duplicated plasmids, which are located close to the cell centre, and induces polymerisation of the ParM actin homolog [C. crescentus MreB has been shown to also affect cell shape and the localization of cell wall synthesizing proteins [B. subtilis are highly dynamic. MreB and Mbl move along helical tracks, with a speed of about 0.1 μm/s, providing potential motor like force [During the depletion of n defect ,9, likew E. coli . It is u E. coli , which i E. coli . Plasmid homolog . ParM fi homolog . On the proteins , and to proteins -16, whicke force . Actin pBacterial chromosome segregation is a highly organized process, depending on several essential protein complexes. DNA polymerase localizes to the cell centre throughout most of the cell cycle . During B. subtilis MreC and MreD proteins localize to the cell membrane, and affect the localization of MreB, likewise to Mbl and MreBH, showing that an intricate interplay exists between actin orthologs and MreCD membrane proteins.We have investigated the role of MreB and Mbl in the positioning of the replication factory, and investigated the role of other actin like proteins and membrane proteins for the localization of MreB. We have found that mreB may also lead to the depletion of MreC, because the mreC gene lies directly downstream of mreB, so, it was unknown if depletion of MreB also affects cell shape directly, and if so, to which extent, or if the observed defect in cell shape is due to a polar effect on mreC. Since both genes are essential [mreD gene lies downstream of mreC, and loss of MreD also leads to a defect in cell shape [mreCD at an ectopic site on the chromosome, which fully complemented the loss of the original mreCD genes. Depletion of mreB during continued synthesis of mreCD from the ectopic site resulted in cessation of growth . We intB. subtilis and in E. coli cells. To investigate a possible role of actin like proteins in this positioning, we depleted MreB or Mbl in cells expressing DnaX-CFP, the tau subunit of the replication DNA polymerase core machinery. In wild type cells showing clear foci (92%), 67% contained a single focus that was positioned close to the cell centre , whereas in 32% of the cells, the DnaX-CFP signals were more than 0.4 μm away from the cell centre Fig. . NeverthTo obtain a more detailed view on the nature of the helical MreB filaments, Z sections were taken through the cells, and 3D image reconstruction was performed on the stacks of fluorescent images. Fig. spo0J mutant cells, which have slightly decondensed DNA, or into smc mutant cells, in which the nucleoids are highly decondensed, and which contain less negatively supercoiled DNA compared to wild type cells [spo0J mutant cells (data not shown), and highly aberrant elongated filaments throughout smc mutant cells .MreB is upstream of mreC and mreD genes, whose depletion leads to formation of round cells (see above, [e above, ,22,26). gfp-mreB copy to the amylase locus under control of the hyperspank promoter that is induced by IPTG, while mbl, mreBH, mreC or mreD genes were driven by the xylose promoter that is induced by xylose (in fructose medium), and is repressed in glucose medium lacking xylose. GFP-MreB filaments were observed in 85–90% of exponentially growing cells in the presence of IPTG .B. subtilis actin ortholog MreB. Our experiments establish that MreB has a dual function, it is vital for the formation of proper rod shape of the cells, and for regular chromosome segregation. However, its function in cell shape is different from that of MreC, or of MreD. The depletion of MreC and MreD leads to rapid cessation of growth and to the formation of small round cells, whereas the sole depletion of MreB results in the formation of large oval shaped cells, and a slower occurring growth arrest. Interestingly, though, we found a connection between MreC and MreB, because during depletion of MreCD, MreB formed fewer and usually abnormally shaped helical filaments. Similar observations have recently been made in E. coli cells [B. subtilis membrane, establishing a link between MreB and the membrane. We speculate that MreC and MreD might provide low affinity binding sites for MreB, such that the filaments extend underneath the membrane in a regular helical pattern. Our results also suggest a dual function for MreC, because its deletion affects the localization of MreB (which is apparently not severe enough to strongly interfere with chromosome segregation), as well as cell shape (in a manner distinct from MreB).This work provides several important conclusions on the function and localization of the li cells . Our expB. subtilis cells. Soon after the depletion of MreB, the replisome lost its central position in the cell, before a change in cell shape was apparent. The depletion of Mbl had only a minor effect on the localization of the replisome, showing that MreB also affects the positioning of an intracellular protein assembly. Our results do not distinguish between the possibilities that the lack of MreB activity results in the loss of central localization of the replisome, which in turn leads to a segregation defect, or that a more direct defect in chromosome segregation due to the lack of MreB might cause mislocalization of the replisome. However, it is tempting to speculate that MreB could actively push DNA away from the central replisome towards opposite cell poles, and that the net result of this simultaneous pushing of ejected DNA towards opposite directions might lead to a balanced positioning of the replisome towards the cell centre, without any need for an anchor. This is in agreement with recent data showing that the replication machinery is highly mobile around the cell centre [An important, if not crucial function of MreB is the positioning of the replication machinery in l centre ,30.An intriguing property of bacterial actin orthologs is the formation of highly dynamic helical filaments underneath the cell membrane that for some members of this protein family are thought to extend through the entire cell length ,16. Thresmc mutant cells, MreB filaments are abnormally spaced and extended, and to a much lesser extent in spo0J mutant cells. This further supports and extends our earlier findings that a connection exists between MreB and the nucleoids. Interestingly, smc mutant cells are elongated and frequently twisted and wider than wild type cells [We also provide evidence that the formation of MreB filaments is affected by the nature of the nucleoids, and by the other actin like proteins. In pe cells , which mOur findings show that an intricate interplay exists between MreB, membrane associated MreC and MreD proteins, other actin orthologs, the replication machinery and the nucleoids, shedding light on the question why the depletion of MreB affects both, chromosome segregation and cell shape. What remains to be investigated are several important questions, e.g. what is the mode of interaction between MreB and the MreCD proteins or with Mbl and MreBH, and to identify the link between MreB and the nucleoids or the replisome, to distinguish between the causality of defects caused by the loss of MreB activity. Also, it will be highly revealing to identify the possible load MreB might be pushing, if its dynamic movement indeed constitutes a motor function within the prokaryotic cell.Escherichia coli XL1-Blue (Stratagene) or B. subtilis strains were grown in Luria-Bertani (LB) rich medium supplemented with 50 μg/ml ampicillin or other antibiotics, where appropriate. For induction of the hyperspank promoter, the culture media were supplemented with 0.1 to 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). For induction of xylose promotor, glucose in S750 medium was exchanged for 0.5% fructose and xylose was added up to 0.5%.Gfp mut1 including MCS was amplified from pSG1729 [gfp mut1 for C-terminal fusion had been excised using KpnI and SpeI. The resulting plasmid pHJDS1 was used to generate N-terminal GFP fusions at the original gene locus. To obtain inducible N-terminal GFP fusion alleles of mreC or of mreD at the original locus, the 5' prime regions (350 to 500 bp) of the genes were PCR amplified and inserted in the EcoRI and ApaI sites of plasmid pHJDS1 to generate pJS14 or pJS15, respectively. To create a fusion of GFP to the N-terminus of mreB, mreC or mreD at an ectopic site on the chromosome, the entire sequences of theses genes were PCR amplified and inserted into the EcoRI and ApaI sites of plasmid pSG1729 [gfp-mreB (pJS22) or of mreCD (pJS23) at the amylase locus, gfp-mreB was PCR amplified from pJS17 and mreCD from B. subtilis PY79 chromosomal DNA, and the products were cloned as HindIII-SphI or as SalI-SphI fragments, respectively, immediately downstream of the hyperspank promotor in plasmid pDR111 . pSG1729  and was  pSG1729  in which pSG1729  to generBacillus, pJS14 or pJS15 plasmids were transformed into wild type B. subtilis (PY79) selecting for chloramphenicol resistance (Cm, 5 μg/ml) to generate strains JS14 (Pxyl-gfp-mreC) or JS15 (Pxyl-gfp-mreD), respectively. For GFP N-terminal fusions at the amy locus, plasmids pJS19 and pJS20 for mreC and mreD were transformed into PY79 selecting for spectinomycin resistance (spec, 25 μg/ml) to generate strains JS19 (Pxyl-gfp-mreC::amy) and JS20 (Pxyl-gfp-mreD::amy), respectively. Strain JS32, in which mreB can be depleted in the presence or absence of mreCD, was created by transforming compentent JS1 cells with chromosomal DNA of JS32. To examine the subcellular localization of GFP-MreB in spo0J or in smc null cells, strain JS19 was transformed with chromosomal DNA from strains AG1468 [Pxyl-gfp-mreB::amy, ΔspoOJ) and JS24 (Pxy-gfp-mreB::amy, smc::kan) respectively. To be able to visualize the localisation patterns of labelled MreB helices in cells depleted of MreC, MreD, Mbl and MreBH cells, chromosomal DNA from strains JS3 (Pxyl-mreC), JS4 (Pxyl-mreD), JS2 (Pxyl-mbl) and JS5 (Pxyl-mreBH) was used to transform strain JS25 (Phyperspank-gfp-mreB::amy)selecting for Cm and spec, generating strains JS29 (Phyperspank-gfp-mreB::amy, Pxyl-mreC), JS30 (Phypespank-gfp-mreB::amy, Pxyl-mreD), JS28 (Phyperspank-gfp-mreB::amy, Pxyl-mbl), and JS31 (Phyperspank-gfp-mreB::amy, Pxyl-mreBH). To express DnaX-CFP in MreB or Mbl depleted cells, chromosomal DNA from JS1 and JS2 was used to transform PG24 competent cells.To express GFP-MreC or GFP-MreD at their original locus in s AG1468  or PGΔ38ce spec,  μg/ml toBacillus strains were grown in S750 defined medium [35] complemented with 1% casamino acids. Fluorescence microscopy was performed on an Olympus AX70 microscope. Cells were mounted on agarose gel pads containing S750 growth medium on object slides. Images were acquired with a digital CCD camera; signal intensities and cell length were measured using the Metamorph 4.6 program . For and 3D reconstruction, 10 to 12 images (spacing between 0.2 to 0.38 μm) were taken through the focal plane, and processed in Metamorph 6 program. DNA was stained with 4',6-diamidino- 2-phenylindole  and membranes were stained with FM4-64 .For microscopic analysis, H J D S performed all experiments, PLG helped with 3D image reconstructions, conceived the study, and participated in its design and coordination. All authors read and approved the final manuscript.


We correlated p53 protein expression with pathological subtype and clinical outcome in 75 embryonal brain tumors. The presence of JC virus, which results in p53 protein accumulation, was also examined.p53 protein levels were evaluated semi-quantitatively in 64 medulloblastomas, 3 atypical teratoid rhabdoid tumors (ATRT), and 8 supratentorial primitive neuroectodermal tumors (sPNET) using immunohistochemistry. JC viral sequences were analyzed in DNA extracted from 33 frozen medulloblastoma and PNET samples using quantitative polymerase chain reaction.p53 expression was detected in 18% of non-anaplastic medulloblastomas, 45% of anaplastic medulloblastomas, 67% of ATRT, and 88% of sPNET. The increased p53 immunoreactivity in anaplastic medulloblastoma, ATRT, and sPNET was statistically significant. Log rank analysis of clinical outcome revealed significantly shorter survival in patients with p53 immunopositive embryonal tumors. No JC virus was identified in the embryonal brain tumor samples, while an endogenous human retrovirus (ERV-3) was readily detected.Immunoreactivity for p53 protein is more common in anaplastic medulloblastomas, ATRT and sPNET than in non-anaplastic tumors, and is associated with worse clinical outcomes. However, JC virus infection is not responsible for increased levels of p53 protein. INI1 mutations, and cause particularly grim clinical outcomes [The current World Health Organization classification for tumors of the nervous system includes medulloblastoma, medulloepithelioma, ependymoblastoma, supratentorial primitive neuroectodermal tumor (sPNET) and atypical teratoid/rhabdoid tumor (ATRT) in the category of embryonal brain neoplasms . These toutcomes . Medullooutcomes . Global outcomes .Little is known about the differences in p53 expression and function among the various embryonal brain tumor subtypes. Initial reports on the p53 tumor suppressor gene suggested it was mutated in 10% or less of medulloblastomas . HoweverSeveral other lines of evidence also support a role for the p53 pathway in medulloblastomas. First, medulloblastomas sometimes arise in the context of Li Fraumeni syndrome, in which p53 germline mutations predispose patients to a wide range of neoplasms . Second,It has been suggested that viral infection of human CNS tissues could promote formation of brain tumors by inhibiting p53 and Rb activity . Some reIn order to confirm the association between p53 immunopositivity, clinical outcome, and embryonal tumor subtype, we stained a tissue array containing representative cores from 80 embryonal brain tumors for p53. We also investigated JC virus infection as a possible mechanism for accumulation of p53 protein by searching for viral sequences using a highly sensitive quantitative real time polymerase chain reaction (PCR) assay. We found an association between p53 immunoreactivity, clinical outcome, and tumor subtype, but did not detect JC virus in medulloblastoma or supratentorial PNET.Medulloblastomas and other embryonal brain tumors diagnosed at the Johns Hopkins University Department of Pathology were identified through review of departmental records. Classic, desmoplastic/nodular and large cell/anaplastic medulloblastomas were classified using World Health Organization guidelines . NuclearThe tissue array was sectioned at four microns, deparaffinized, and subjected to antigen retrieval by steaming (20 minutes at 80°C). Slides were then incubated at room temperature for 45 minutes with monoclonal antibody directed towards p53 (1:2000, clone DO-7, DAKO, Carpinteria, CA). Primary antibody was detected using the avidin-biotin complex (ABC) method with diaminobenzadine serving as the chromagen. We semiquantitatively graded staining intensity as negative, weak, or strong. Carcinomas with mutations leading to p53 stabilization were used as positive controls. No staining was seen in the absence of primary antibody (negative control).JC virus sequences were amplified from DNA using the forward primer PEP-1 (5'-AGT CTT TAG GGT CTT CTA CC-3') and reverse primer PEP-2 (5'-GCC AAC CTA TGG AAC AG-3') . Additio1/N was used to calculate the fiducial (exact) 95% upper bound of JC virus prevalence.p53 statistical analyses were performed using GraphPad PRISM4 software (GraphPad Software, San Diego, CA). A two tailed Fishers Exact test was used to compare immunohistochemical staining profiles between groups. Significance of survival differences was assessed using log-rank analysis of Kaplan-Meier curves. The formula 1-We used immunohistochemistry to examine p53 protein expression in representative cores from 80 embryonal brain tumors on a tissue array. Cores from 5 of the cases could not be evaluated due to crush artefact, cautery, or low cellularity. Of the remaining 75 tumors, 64 were medulloblastomas; 31 of these medulloblastoma were of the classic subtype, 13 were nodular/desmoplastic, and 20 were large cell/anaplastic. The tissue array used in this study also contained 11 other CNS embryonal tumors, including 8 sPNET and 3 ATRT. Among the sPNET were 3 with the long epithelial surfaces characteristic of medulloepithelioma.Immunoreactivity for p53 was present in a minority (35%) of the 75 embryonal tumors, and a relatively small subset of cells stained in most of the positive cases. In all positive cases the majority of the immunoreactivity was in tumor cell nuclei. An example of a nodular medulloblastoma with weak, scattered p53 immunoreactivity is shown in Figure We next examined whether p53 immunopositivity was associated with worse clinical outcomes in embryonal brain tumor patients. Of the 75 tumors from 73 patients scored on the array, survival data was available for 66 individuals. 61 percent of patients were alive at last contact, with follow up times ranging from 3 to 215 months (median 47 months). When survival of the entire embryonal brain tumor cohort was analyzed, 67% (31/46) of patients with p53 immunonegative tumors were alive, as compared to 45% (9/20) of patients with immunopositive tumors. Log rank analysis of Kaplan Meier survival curves confirmed the significance of this difference (P = 0.02). When only the 56 medulloblastoma patients with clinical follow up were analyzed, 71% of individuals with p53 negative tumors survived, compared to 58% of individuals with p53 positive tumors. However, these differences were not significant on log rank analysis (P = 0.25). The 6 patients with intensely immunoreactive tumors (3 anaplastic medulloblastoma, 2 sPNET, 1 ATRT) all died from their disease in less than two years.3 - 2 × 105 per PCR assay (mean 3.4 × 104). Because two copies of ERV-3 are present per human genome, DNA from at least 1000 cells was assayed in each PCR reaction. In contrast, all of the specimens tested were completely negative for JC virus. The quantitative Taqman assay was able to detect a minimum of ~10 copies of JC virus per PCR reaction as determined using a standard dilution curve. Thus the number of JC virus genomes should not exceed 10 per 1,000 tumor cells. Given our 0% detection rate, the 95% confidence limit for the largest value of the 'true' underlying JC virus prevalence is no more than 7.6% (1-(0.05)1/38).To address the possibility that p53 accumulation in medulloblastoma and other embryonal brain tumors is due to viral infection, we used quantitative RT PCR to search for viral sequences in tumor DNA. Of the cases used on the tissue array, 19 had material frozen suitable for high-quality DNA extraction. Four of these cases were p53 positive. Also available in our frozen tumor bank was tissue from 14 additional embryonal lesions (4 classic medulloblastoma, 5 anaplastic medulloblastoma, 1 nodular medulloblastoma and 4 sPNET). We did not detect JC virus sequences in any of these 33 tumors. We repeated the analysis on DNA extracted from a different tumor tissue fragment in 5 cases, but these spatially distinct regions also failed to contain viral DNA. ERV-3, an endogenous human retroviral element, served as a control and was easily amplified from tumor DNA samples  as compared to non-anaplastic ones (18% positive). The percentage of p53 immunopositive medulloblastomas in our study (27%) fell within the previously reported range of 3% to 53% ,17,33,34Interestingly, supratentorial PNET and ATRT were also more commonly p53 immunopositive than non-anaplastic medulloblastoma in our study. While extracerebellar PNET were included in several earlier studies, p53 immunoreactivity was not reported separately for these lesions ,15. Ho aClinical outcomes were significantly worse for embryonal brain tumor patients in our study whose lesions were p53 immunopositive. However, cases most commonly positive for p53 (anaplastic medulloblastomas, sPNET, and ATRT) are all more clinically aggressive than non-anaplastic medulloblastomas, making it difficult to infer causality resulting from p53 accumulation. p53 expression did not predict outcome within the group of medulloblastoma patients, although all 3 strongly p53 immunopositive tumors were severely anaplastic and associated with quite short survival. Interestingly, in a recently published report, Ray and colleagues found that p53 immunoreactivity was the only biological marker predictive of poor outcome on both univariate and multivariate analyses in a group of 112 medulloblastoma patients .We also examined the potential role of viral infection as a mechanism for p53 protein stabilization in medulloblastoma. None of the four p53-positive cases from our tissue array with frozen material available contained viral sequences. We also tested nine additional samples of histological subtypes that were more commonly associated with p53 immunopositive staining (anaplastic medulloblastomas and sPNETs) where p53 staining was not performed. None of these tumors were JCV positive. While sample availability precluded complete testing of all confirmed p53 positive tumors for JCV, the available data do not support a causal role for JCV infection in p53 accumulation and development of embryonal brain tumors.The impact of viruses on medulloblastoma pathogenesis is controversial. The most commonly implicated agents are the polyomavirus family members JC virus, BK virus and SV40 virus. Of these, JC virus, which infects approximately 80% of the pediatric population and can cause CNS disease in immunosuppressed patients, has been most studied. It was shown decades ago that inoculation of JC virus into rodents resulted in formation of medulloblastoma-like cerebellar tumors ,22,42,43We failed to detect JC virus sequences in the 33 cases examined, using a sensitive and specific technique which should identify as few as 10 viral genomes per PCR reaction. This suggests the p53 immunopositivity we observe is not caused by JC virus infection in the majority of cases. Our data are also not consistent with the hypothesis that ongoing JC virus infection is common in medulloblastoma. Other recent studies have also reported a lack of JC virus DNA in medulloblastomas. Hayashi and colleagues failed to identify JC viral sequences in 13 medulloblastomas . Kim andIn summary, we find significantly increased p53 protein levels in anaplastic medulloblastomas, sPNET, and ATRT as compared to classic and nodular medulloblastoma. JC virus was not detected in the 33 tumors examined, suggesting that T-antigen binding does not appear to be an ongoing factor in the pathobiology or p53 protein accumulation of embryonal brain tumors.The author(s) declare that they have no competing interests.Drs. Eberhart, Shah and Gravitt planned the study and wrote the initial manuscript draft. Dr. Eberhart, Ms. Chaudhry and Ms. Khaki collected samples, isolated DNA, and performed immunohistochemical staining. Dr. Gravitt supervised and R Daniel performed the JC virus copy number analysis in tumors. All authors reviewed and commented on the final manuscript.The pre-publication history for this paper can be accessed here:


The Angiotensin Converting Enzyme (ACE) insertion/deletion (I/D) polymorphism has received much attention in pharmacogenetic research because observed variations in response to ACE inhibitors might be associated with this polymorphism. Pharmacogenetic testing raises the hope to individualise ACE inhibitor therapy in order to optimise its effectiveness and to reduce adverse effects for genetically different subgroups. However, the extent of its effect modification in patients treated with ACE inhibitors remains inconclusive. Therefore our objective is to quantify the effect modification of the insertion/deletion polymorphism of the angiotensin converting enzyme gene on any surrogate and clinically relevant parameters in patients with cardiovascular diseases, diabetes, renal transplantation and/or renal failure.Systematic Review. We will perform literature searches in six electronic databases to identify randomised controlled trials comparing the effectiveness and occurrence of adverse events of ACE inhibitor therapy against placebo or any active treatment stratified by the I/D gene polymorphism. In addition, authors of trials, experts in pharmacogenetics and pharmaceutical companies will be contacted for further published or unpublished data. Hand searching will be accomplished by reviewing the reference lists of all included studies. The methodological quality of included papers will be assessed. Data analyses will be performed in clinically and methodologically cogent subgroups. The results of the quantitative assessment will be pooled statistically where appropriate to produce an estimate of the differences in the effect of ACE inhibitors observed between the three ACE genotypes.This protocol describes a strategy to quantify the effect modification of the ACE polymorphism on ACE inhibitors in relevant clinical domains using meta-epidemiological research methods. The results may provide evidence for the usefulness of pharmacogenetic testing for individualised ACE inhibitor therapy. Research in genetics and genome sequencing has led to a better understanding of the molecular genetic mechanisms and to the detection of inter-individual genetic differences, so-called polymorphisms, which may have a functional consequence on the response to drugs. Pharmacogenetic tests provide information to better predict and prevent therapeutic failures and adverse drug reaction and raise the hope for an individualised pharmacotherapy -3. AlthoThe ACE polymorphism identified in 1990 by Rigat and co-workers  is one oTherefore, our objective is to systematically review all randomised controlled trials that assessed to what extent the insertion/deletion polymorphism of the angiotensin converting enzyme gene influences the effect and adverse events of angiotensin converting enzyme inhibitors on any surrogate and clinically relevant parameters in patients with cardiovascular disease, diabetes, renal transplantation and/or renal failure.We will perform literature searches in (Pre-) MEDLINE (DataStar version, Cary North Carolina), EMBASE (DataStar version, Cary North Carolina), Biosis (Ovid version "Previews 1989 to 2003", New York, New York), the Cochrane Controlled Trials Register (CCTR <3rd Quarter 2003>, Oxford, United Kingdom) and the Science citation index. A preliminary literature search in Medline has been carried out to estimate the range of relevant literature. Out of the citations of the pilot searches (172 citations) we identified articles that met our inclusion criteria. Keywords of these articles were used to refine our search strategies. In collaboration with an information specialist we designed the final search strategies for the six databases avoiding any language restrictions [see In addition, authors of trials identified in the literature search will be contacted for additional published or unpublished data. Particular efforts will be made to obtain unpublished data on genetic test information and effect measures stratified according to the genetic subtypes examined. We will send our requests and subsequent reminders for additional data to the first and last authors.Other contacts will include the relevant collaborative review groups of the Cochrane Collaboration, pharmaceutical companies and manufacturers and researchers known to have published pharmacogenetic analyses in the area of cardiovascular disease, diabetes, renal transplantation and/or renal failure. Hand searching will be accomplished by reviewing the bibliographies of all included studies to identify additional relevant articles as well as by using the "related articles" function of PubMed and the citation index of ISI Web of Science.Anticipating that subgroup analyses investigating gene polymorphisms may not be specifically mentioned in titles or abstracts, we will study the full text of all randomised placebo-controlled trials (RCT) that assessed the effectiveness of ACE inhibitors in order to identify subgroup analyses investigating gene polymorphisms.Two reviewers will independently assess all obtained titles and abstracts of the literature search for inclusion. The criteria to be used to identify relevant studies will be 1) randomised controlled trials 2) the investigation of an angiotensin converting enzyme inhibitor used for one of the clinical domains mentioned below and 3) the determination of the deletion/insertion polymorphism of patients.The two reviewers will then examine the full texts of all potentially relevant citations. The decision on in- and exclusion will be based on the following, more explicit inclusion criteria.We will include studies investigating ACE inhibitors in the four major clinical domains namely cardiovascular diseases, diabetes, renal transplantation and/or renal failure.Studies should include patients with the following indications for ACE inhibitor therapy: Heart failure, primary and secondary hypertension, coronary artery disease, diabetic nephropathy, primary nephropathy and status after renal transplantation.All licensed or unlicensed ACE inhibitors identified through the literature search will be included.We will prefer placebo as control intervention in order to study the effect modification. However to assess all available evidence, we will also include pragmatic trials where patients with active treatments  served as controls.We are mainly interested in studies investigating a single drug exposure with ACE-inhibitors. However we will also include those studies, which allowed co-medications.Studies must include a description on how determination of the angiotensin converting enzymes genotypes (DD/ DI/ II) has been performed. If a study does not report details of testing but provides relevant results, authors will be contacted to obtain information of testing technique.We will secure data on any reported outcome, surrogate endpoints  and clinically relevant outcomes ).The two reviewers will resolve any discrepancies about in- or exclusion by discussion. If agreement cannot be achieved, a third reviewer will make the decision.We will use a pre-designed data extraction form that includes different items to assess the studies' external validity [see All trials included in the review will be assessed using a list of selected quality items indicating components of internal validity and descriptive information . In prinIn addition, we will assess the description of the methods to determine genotypes. Angiotensin converting enzymes genotypes (DD/ DI/ II) are traditionally determined using polymerase chain reaction (PCR) amplification according to previously published protocols . The D aWe will pre-test these quality assessment items on a small sample of studies in duplicate and if necessary add missing descriptive items.Two reviewers will independently score the internal and descriptive validity. The initial degree of discordance between the reviewers will be reported. Discordant scores based on obvious reading errors will be corrected. Discordant scores based on real differences in interpretation will be resolved through consensus. A third party will be sought if necessary. The reviewers will not be blinded for names of authors, institutions, journals or the outcomes of the studies.These detailed quality assessment will be used to describe the methodological quality of selected studies, to explore potential sources of heterogeneity, to make informed decisions regarding suitability of meta-analysis and to weigh the strength of any conclusions.The results of the data extraction and assessment of study validity will be presented in different structured tables and in a narrative description [see Finally the tables will provide the individual study results  of the different genotypes in the intervention and control group. Continuous outcomes (e.g. blood pressure) will be summarised in the table as mean differences between baseline and follow up measures. For data of dichotomous outcomes (e.g. cardiovascular death) the relative risks between the results of the DD genotype and the II genotype will be calculated and described in the table. A relative risk of one indicates no difference between two genotypes, where as relative risks lower respectively higher than 1 indicates variations in the treatment effect.The heterogeneity assessments help us to examine study characteristics that might be related to variability in the observed outcome. Within each subgroup potential sources of heterogeneity that may affect the imprecision in the estimate of treatment effect such as the study methodology, population characteristics, intensity of intervention, co-medications and risk factors will be examined. We will perform multiple linear regression analyses (meta-regression) to explore sources of between-study heterogeneity. The log transformed odds ratio for dichotomous outcomes  and continuous outcomes (blood pressure) measurements will be used as dependent variables and the clinical and methodological items of the extraction sheet as described above will be entered into the model as independent variables.When a factor is strongly associated with the variation in ln odds ratio or on the continuous outcome, we will stratify the studies on that variable and inspect residual heterogeneity using forest plots.If a meta-analysis seems appropriate, that is when the p-value of the chi-squared test for heterogeneity is greater than 0.10, a fixed effects model will be used for pooling. Within clinically and methodologically cogent subgroups relative risks for dichotomous outcomes and weighted mean differences for continuous outcomes will be calculated comparing the contrast between the intervention and the control group within genotypes. The results between the different genotypes will be presented in a forest plot as shown in Figure The pooled results of a cogent subgroup will produce an estimate of the differences in the average effect of ACE inhibitors observed between ACE genotypes. Thus the treatment effect of each genotype could be compared to the overall effect of ACE inhibitors regardless of the genotype.® Statistical Software: Release 8.2 College Station, Texas, USA).All statistical analyses will be performed using the Stata statistical software package  on different outcomes and in the light of study methodology and participants characteristics. These results should inform clinicians about the potential of pharmacogenetic testing to individualise ACE inhibitor treatment.None declared.LMB, JS and MS initiated the project. MS wrote the first draft of the protocol. MP, JS and LMB critically reviewed and revised the manuscript. All authors approved the manuscript.The pre-publication history for this paper can be accessed here:Search strategies: Search terms and number of citations listed for five electronic databasesClick here for fileData extraction and quality assessment sheet: Example of data extraction and quality assessment for the study of Hernandez [21]Click here for file


Fragility fractures caused by osteoporosis are a major cause of morbidity and mortality in aging populations. Bone mineral density (BMD) is a useful surrogate marker for risk of fracture and is a highly heritable trait. The genetic variants underlying this genetic contribution are largely unknown.2, n = 319) and high (> 1.11 g/cm2, n = 321) BMD at the lumbar spine. Significant findings were verified in two additional sample collections.We performed a large-scale association study investigating more than 25,000 single nucleotide polymorphisms (SNPs) located within 16,000 genes. Allele frequencies were estimated in contrasting DNA pools from white females selected for low (<0.87 g/cmPDE4D) gene region on chromosome 5q12. We subsequently tested the marker SNP, rs1498608, in a second sample of 138 white females with low (<0.91 g/cm2) and 138 females with high (>1.04 g/cm2) lumbar spine BMD. Odds ratios were 1.5 (P = 0.035) in the original sample and 2.1 (P = 0.018) in the replication sample. Association fine mapping with 80 SNPs located within 50 kilobases of the marker SNP identified a 20 kilobase region of association containing exon 6 of PDE4D. In a second, family-based replication sample with a preponderance of females with low BMD, rs1498608 showed an opposite relationship with BMD at different sites (p = 0.00044-0.09). We also replicated the previously reported association of the Ser37Ala polymorphism in BMP2, known to interact biologically with PDE4D, with BMD.Based on allele frequency differences between DNA pools and subsequent individual genotyping, one of the candidate loci indicated was the phosphodiesterase 4D (PDE4 gene family in human osteoporosis.This study indicates that variants in the gene encoding PDE4D account for some of the genetic contribution to bone mineral density variation in humans. The contrasting results from different samples indicate that the effect may be context-dependent. PDE4 inhibitors have been shown to increase bone mass in normal and osteopenic mice, but up until now there have been no reports implicating any member of the  COL1A1) gene has shown one of the most consistent associations to osteoporosis, even if the association is generally weak for BMD and varies between populations [BMP2, the gene encoding for bone morphogenetic protein 2, as responsible for the linkage [The postmenopausal loss of bone mass and subsequent increased risk of low-energy (fragility) fractures is an important public health problem, especially in countries with a high proportion of elderly individuals. More than 1 million fragility fractures, primarily in postmenopausal women, occur each year in the US. The annual direct medical costs exceed US$10 billion -5. Thereulations -11. Linkulations -19. So f linkage . NeverthPDE4D, the gene encoding cyclic AMP-dependent phosphodiesterase 4D, with lumbar spine BMD. PDE4D selective inhibitors have been shown to promote osteoblast differentiation in progenitor cells [Studies that rely on direct association approaches based on linkage disequilibrium within populations are expected to have greater statistical power and be more feasible to implement than traditional linkage studies to identify common variations that influence common, complex traits such as osteoporosis . Recentlor cells  and to ior cells  but the The population sample from which the discovery samples were chosen consisted of 5,436 female twins collected at the Twin Unit at St Thomas Hospital, London, England. They were selected without regard to health or trait. The volunteers had been recruited through advertisements and had undergone extensive investigation at the Twin Unit at St Thomas Hospital. Investigations included several questionnaires inquiring about present and past diseases, symptoms, family history, socio-economical factors, and medication. Subjects underwent an extensive clinical assessment including DEXA measurements of bone mineral density and anthropometric measurements ,24. All A twin sample from Royal North Shore Hospital, Sidney, Australia was collected similarly as the UK twin collection. 731 individuals including twin pairs and singletons with lumbar spine BMD assessments were available for genotyping  study that collected sib pairs concordant and discordant for bone mineral density . ProbandAll studies were approved by the appropriate research ethics committees. All participants gave their informed consent to participate in genetic studies before enrollment.Bone mineral density was estimated from the L1-L4 vertebrae, hip, and forearm using DEXA according to the user's manual for the Hologic QDR 4500W,  at all collection sites.A set of 25,494 SNP markers was selected from a collection of 125,799 experimentally validated polymorphic variations [For pooled DNA assays, 25 ng of case and control DNA pools was used for amplification at each site. All PCR and MassEXTEND™ reactions were conducted using standard conditions . RelativBMP2 were AGCTGGGCCGCAGGAAGTTCG (forward PCR primer), TCGTCAGAGGGCTGGGATGAG (reverse PCR primer) and TGAGGGGCGGCCCGACG (extension primer).Primers used to genotype rs1498608 were ATAACCTCGGGGTCCAGAAA (forward PCR primer), GAATCCCTGTTCATTCCTTG (reverse PCR primer) and CCCTAAAAACTGTTCCAGGTA (extension primer). The primers used to genotype the Ser37Ala polymorphism in -5, PCR/mass extension = 1.7 × 10-4, and chip measurement = 1.0 × 10-4.Tests of association between adjusted lumbar spine BMD group and each SNP using pooled DNA were carried out in a similar fashion as explained elsewhere . BrieflyTests of association using individual genotypes were carried out using a chi-square test of heterogeneity to compare allele frequencies, and Fisher's exact test to compare genotype frequencies (due to low frequency contingency table cells). Confidence intervals and P-values for odds ratios were derived using Fisher's exact test. When one or more cell counts were zero, non-infinite odds ratios were estimated by adding 0.5 to each cell . In the 2 = 0.82) [2 in the low BMD group and 1.11–1.60 g/cm2 in the high BMD group, corresponding to the upper and lower 22nd percentiles. Other characteristics of the samples are described in Table We carried out a genome-wide association study using 25,494 SNPs located within 10 kb of 15,995 LocusLink annotated genes. An overview of the investigative process is shown in Figure  = 0.82) . The selPDE4D on chromosome 5q12. Allele frequencies for the T allele based on genotyping were 0.91 in the low lumbar spine BMD pool and 0.88 in the high BMD pool (OR = 1.5, P = 0.035). Complete genotype counts and summary statistics are reported in Table One of the associations was found with rs1498608, an A/T polymorphism within intron 5 of Genome-wide studies using tens of thousands of SNPs and liberal statistical selection criteria are expected to yield a high proportion of false positive associations. To distinguish the true genetic effects from among the false positives, the 78 selected SNPs were genotyped in a second twin sample from Australia.2, P = 0.049) or TT (β = 8.0 g/cm2, P = 0.037) genotypes, thus confirming the results observed in the unrelated tails of this sample. Similar GEE analyses carried out for femoral neck and hip BMD were not statistically significant.The Australian replication sample, a combination of female and male twin pairs and singletons, was analyzed in two ways. First, to create a design and carry out an analysis comparable to the discovery sample, unrelated individuals were selected from the lower and upper quartiles of the sex-specific adjusted lumbar spine BMD distribution Table . A simil2, P = 0.11) and the TT (β = -5.4 g/cm2, P = 0.09) genotypes. Using Z-scores at the femoral neck (P = 0.0007 and 0.0004), total hip (P = 0.003 and 0.007), and lumbar spine (P = 0.03 and 0.02) as dependent variables confirmed this pattern of association. In all cases the AT and TT genotypes had very similar point estimates.Being a sample of mostly affected sib pairs, this sample was unsuitable for formation of groups with contrasting BMD because of the preponderance of individuals with low BMD Table . TherefoPDE4D.In order to better define the extent of the region of association and possibly identify other SNPs more strongly associated with BMD, we performed DNA pool based association fine mapping in the UK sample using 80 publicly available intronic SNPs in the 100 kb region surrounding the incident SNP Figure . This an2 (P = 0.0029). There were no homozygous Ala individuals in this sample.As described in the discussion below, PDE4D inhibition is known to influence BMP2-induced alkaline phosphatase activity in osteoblast precursor cells. Recently, variation in the gene encoding BMP2 was found to be associated with osteoporosis in a study employing whole genome linkage and subsequent positional cloning . Since wPDE4D is associated with low bone mineral density at the lumbar spine in females. PDE4D encodes cyclic AMP-dependent phosphodiesterase 4D. Phosphodiesterases are a superfamily of enzymes involved in degradation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) [PDE4 genes in human osteoporosis. However, variation in PDE4D was recently reported to be associated with the risk of ischemic stroke [PDE4D variants may have effects on the risk for different common diseases. There are other examples of genes having such pleiotropic effects, the most notable being APOE in hyperlipidemia and Alzheimer's disease [PDE4D association with stroke was strongest for a region in the recently extended 5' end of the gene, which is close to 1,000 kb upstream of rs1498608 [PDE4D to the risk of osteoporosis as well as stroke, it is possible that different domains are involved in the different diseases.In an association study using SNPs in nearly 16,000 genes we obtained evidence that variation in the SNP rs1498608 located within e (cGMP) ,35. cAMPe (cGMP) -39, whice (cGMP) -45. The e (cGMP) , and dege (cGMP)  and to ae (cGMP) ,48. PDE4e (cGMP) . PDE4 ine (cGMP) . In spit disease ,51. It sBMP2 and measures of osteoporosis in an international family-based sample ascertained via low BMD probands. Although not statistically significant, this finding was supported by the results in the discovery sample of unrelated high and low spine BMD subjects. The allele frequency in the low BMD group was 2.2% and in the high group 1.4%, with an odds ratio of 1.6 (P = 0.28). The rare allele was less common in our low BMD group than the low spine BMD group (3%) in the Icelandic sample. However, our allele frequencies in the low and high BMD groups and the resulting OR corresponded well with the figures in the Danish sample (1.8% vs 1.0%, RR = 1.8) reported in the same paper [BMP2 and PDE4D in either sample. However, given the low minor allele frequencies of each SNP, there was very little power to test for interaction effects.Given the interaction between BMP2 and PDE4 for the inhibition on osteoblastic differentiation in vitro, it is interesting to note that variants in the gene encoding for BMP2 have also been found to increase risk of osteoporosis in humans . In the me paper . We founPDE4D and lumbar spine bone mineral density was detected, providing the first evidence that a variant of this gene could contribute to the risk of osteoporosis in humans. The effect was similar in size in premenopausal and postmenopausal women, indicating that the effect would be on the attainment of peak bone mass rather than the rate of decrease in BMD after menopause. The lack of a detectable interaction with female sex hormones is supported by having observed a similar genetic effect in the small sample of males in our study. The genetic contribution to peak bone mass is possibly bigger and definitely better documented than the as yet unproven genetic influence on postmenopausal bone loss [PDE4D could contribute to this effect, especially in light of the documented anabolic effect on bone by PDE4 inhibitors.The starting point of the present study was a large-scale association study of more than 25,000 SNPs located in 16,000 genes. After a stepwise selection process an association between an intronic SNP in one loss , and it An association with an intronic SNP provides little evidence for a change in amount or function of the protein that could explain the association. None of the 80 SNPs investigated as part of the association fine mapping were non-synonymous coding changes, which is consistent with the extraordinary lack of variation that others have reported for all PDE classes  and PDE4PDE4B in our assay set, rs1318475, was taken through to the second stage . These results suggest that further investigation into possible associations between variants in all PDE4 genes and bone mineral density may be justified.Given the functional similarity between different PDE4 enzymes, we went back and scrutinized our data for associations with SNPs in the other PDE4 genes that may have been overlooked during the first stage of the scan. The only SNP in PDE4D marker and bone mineral density was one result from over 25,000 hypothesis tests. A conservative Bonferroni adjustment to yield an experiment-wide type I error rate of 0.05 would demand a test-wise p-value on the order of 10-6. Given the modest sample size, only common variations with relatively large effects (OR > 2) would reach such significance levels. Instead, we chose to be more mindful of the role of type II error rates and apply a more liberal set of criteria in the initial phases of the study and verify true genetic effects by independent replication. The analysis of 78 selected markers in the Australian replication sample resulted in multiple associations of continuing interest, with rs1498608 displaying one of the strongest associations. A one-sided test of association comparing the results in the discovery and replication samples yields a p-value of 0.0074. This would not be considered significant on an experiment-wide level after Bonferroni adjustment, which would require a p-value on the order of 0.0006 or lower. The analysis in the international replication sample produced contradictory data in that the A allele, which in the first two samples was associated with increased lumbar spine bone mineral density, was associated with decreased BMD at all tested sites. The pattern of association evident from the first two samples, with AT and TT genotypes having very similar point estimates, was preserved in this sample, even in the face of the reverse direction of association. The highly statistically significant association between rs1498608 and femoral neck and hip BMD in this third sample and the consistency in the pattern of association would be unexpected from a spurious result. A possible explanation for the contradictory results could be the fact that the international sample consists mostly of individuals with low BMD since the probands all have a BMD Z-score < -1.5, and most of the siblings also have low BMD. It is possible that within such a selected sample the relationship between rs1498608 and BMD could be altered due to interactions with other genetic or environmental factors.The route by which these genetic associations were arrived at and the potential for spurious association must be considered. Recent published work has brought light to the need for proper validation to verify genetic findings for complex traits -56. In tPDE4D variants in the genetic contribution to BMD in humans merits further investigation.The well-documented anabolic effect on bone by PDE4 inhibitors lends indirect support for the association reported here, and it would seem that the possible role of The result of the present large scale association study together with data from previously published animal models suggest that genetic variation in the gene encoding PDE4D contributes to the variation in lumbar spine BMD in humans.The authors of this manuscript affiliated with Sequenom, Inc. (RR, SM, CH, GM, SK, MN, AB) declare competing financial interests, which may include current or prior receipt of salary and/or stock ownership, as Sequenom, Inc. may be affected financially by the publication of this manuscript.RR drafted the manuscript and participated in study design and data analysis. SM was study project leader and participated in data analysis. SK supervised the operational aspects of the study, and was responsible for the development of the SNP marker assay set. CH and GM participated in the development of the SNP marker assay set. SW was project manager for the international multicenter study. PS was principal investigator for the Australian Twin collection. TS was principal investigator for the UK twin and international collections. MN participated in study design, performed the statistical analyses and helped draft the manuscript. AB had the overall scientific responsibility for the study.The pre-publication history for this paper can be accessed here: