Saccharomyces cerevisiae (Yao, X.-J., J. Lemay, N. Rougeau, M. Clément, S. Kurtz, P. Belhumeur, and E. A. Cohen, J. Biol. Chem. v. 277, p. 48816–48826, 2002). In this study, we performed a mutagenic analysis of Vpr to identify sequence and/or structural determinants implicated in the interaction with di-W-containing peptides and assessed the effect of mutations on Vpr-induced cytostatic activity in S. cerevisiae.The HIV-1 genome encodes a well-conserved accessory gene product, Vpr, that serves multiple functions in the retroviral life cycle, including the enhancement of viral replication in nondividing macrophages, the induction of G2 cell-cycle arrest, and the modulation of HIV-1-induced apoptosis. We previously reported the genetic selection of a panel of di-tryptophan (W)-containing peptides capable of interacting with HIV-1 Vpr and inhibiting its cytostatic activity in S. cerevisiae and remained sensitive to the inhibitory effect of di-W-containing peptides.Our data clearly shows that integrity of N-terminal α-helix I (17–33) and α-helix III (53–83) is crucial for Vpr interaction with di-W-containing peptides as well as for the protein-induced cytostatic effect in budding yeast. Interestingly, several Vpr mutants, mainly in the N- and C-terminal domains, which were previously reported to be defective for cell-cycle arrest or apoptosis in human cells, still displayed a cytostatic activity in Vpr-induced growth arrest in budding yeast can be effectively inhibited by GST-fused di-W peptide through a specific interaction of di-W peptide with Vpr functional domain, which includes α-helix I (17–33) and α-helix III (53–83). Furthermore, the mechanism(s) underlying Vpr-induced cytostatic effect in budding yeast are likely to be distinct from those implicated in cell-cycle alteration and apoptosis in human cells. Increas protein -5. Secon protein .Schizosaccharomyces pombe (Sc. Pombe) [Saccharomyces (S.) cerevisiae [S. Pombe cells [S. cerevisiae has been proposed to result from gross mitochondrial dysfunction [Even though the molecular mechanism of Vpr-mediated cell-cycle G2 arrest is still obscure, it has been known that Vpr expression leads to inactivation of the mitotic p34cdc2/cyclinB complex in human cells ,9 as wel. Pombe) -14. Invo. Pombe) -12,14 burevisiae -17. Delerevisiae ,18. Althbe cells -25, the be cells . Accordibe cells ,27. Indefunction , rather et al. have provided evidence indicating that extracellular Vpr or peptides derived from Vpr C-terminus induce mitochondrial dysfunction in human cells by a mechanism involving a specific binding to the adenine nucleotide translocator (ANT), a component of the permeability transition pore complex (PTPC) in the mitochondrial membrane. The resulting mitochondrial membrane permeabilization (MMP) leads to a decreased membrane potential and the release of cytochrome c and apoptosis inducing factor (AIF) [In addition to nuclear import and cytostatic activities, HIV-1 Vpr exhibits cytotoxic properties. Elevated intracellular expression or addition of extracellular Vpr or derived peptides results in proapoptotic effects in human cells including neurons ,28,29 asor (AIF) ,33. Thisor (AIF) -37. In aS. cerevisiae presumably by interacting with Vpr [S cerevisiae. Results reveal that the inhibitory di-W-containing peptides target specifically a functional domain of Vpr directly involved in growth arrest in budding yeast. Furthermore, several previously well-characterized Vpr mutants unable to induce cell-cycle dysregulation and/or apoptosis in mammalian cells still exhibit strong growth arrest activity in budding yeast, indeed suggesting that Vpr carries out distinct functions in S. cerevisiae.In a previous report, we have shown that expression of genetically-selected glutathione-S-transferase (GST)-fused di-tryptophan (di-W)-containing peptides inhibited Vpr-mediated growth arrest in with Vpr . Intereswith Vpr . Even thS. cerevisiae budding yeast and selected a panel of di-W-containing GST-peptides that specifically inhibit Vpr-mediated yeast growth arrest function presumably through their ability to bind HIV-1 Vpr [We have previously used a genetic selection system in IV-1 Vpr . In thisStructural studies performed with synthetic forms of Vpr indicate that Vpr is characterized by a well-defined gamma turn 14–16)-alpha helix (α-helix I: 17–33)-turn (34–36), followed by an alpha helix(α-helix II: 40–48)-loop (49–54)-alpha helix (α-helix III: 55–83) domain and ends with a very flexible C-terminal arginine-rich sequence . The α-h-alpha heTo determine the impact of the Vpr mutations on GST-B4 peptide binding, HP-16 yeast co-transformed with mutated-Vpr expressors and either GST or GST-B4 vectors were radio-labeled in Vpr-inducible medium and subjected to GST pull-down assays Fig. , as desc-, 2% raf) or a Vpr-inducible plate . Cell growth was evaluated following an incubation of 3–5 days at 30°C , wild-type (R+) or mutated Vpr expressors were serially diluted and spotted onto either a Vpr non-inducible plate  underlying Vpr-induced cytostatic effect in budding yeast are distinct from those implicated in cell-cycle alteration and apoptosis in mammalian cells.We have previously shown that GST-fused di-W-containing peptides were able to interact with HIV-1 Vpr and as a result inhibit its multiple functions in budding yeast as well as in HIV-1 infected T cells . In the S. cerevisiae budding yeast was attributed to its last 63–96 amino acid (aa) and the critical domain was located in a conserved C-terminal HFRIGCRHSRIG sequence from aa 71 to 82 [et al., revealed that substitution mutations of aa His71 or Gly75 in this HFRIG sequence abolished Vpr cytostatic effect in budding yeast [et al showing that the Vpr F34I mutant was unable to induce a growth arrest phenotype in budding yeast [et al [Previous reports have indicated that the Vpr cytostatic activity in 71 to 82 . In contng yeast . Interesng yeast . On the t [et al , mutatiot [et al , and mayin vivo. Such a difference may specifically rely on the hydrophobic di-W motif, which is not present in UDG [One striking observation of this study is that the four mutants (E21K, L23F, A30F and R62P) located in the α-helical I and III regions of Vpr, which were defective for the cytostatic activity in budding yeast  [The rc1-407) . Plasmidrc1-407) .- have been previously described [BamHI site in front of the Vpr initiation codon, a 3'-primer (5'-GCATCGCTCGAGGATCTACTGGC-3') containing a XhoI site after the stop codon of Vpr and the complementary oligonucleotide primers containing the desired mutations. Amplified Vpr cDNA harboring specific mutations were then cloned into the p424Gal1 vector at BamHI/XhoI sites. The Vpr mutants L23F, E25K, A30F, R62P, I63K, R77Q, R77fs and R80A were previously described [S. cerevisiae HP-16 yeast colony that was resistant to HIV-1 Vpr-mediated growth arrest as previously described [The HIV-1 Vpr yeast expression plasmid  and the negative control plasmid p424Gal1-Rescribed . To geneescribed  by usingescribed ,48. The escribed  while thescribed .The rabbit anti-Vpr polyclonal serum was raised against bacterially expressed recombinant Vpr as described previously . Galacto- or Trp-/Ura-, 2% raffinose (raf+)) for 2 days. Then, suspensions of transformed HP-16 yeast cells (adjusted at similar cell densities) were serially diluted and spotted onto either a Vpr non-inducible plate (Trp- or Trp-/Ura-, 2% raf) or a Vpr-inducible plate  to evaluate the growth of each co-transformed HP16 population.The experimental procedures to evaluate protein expression, Vpr growth arrest activity and the anti-Vpr activity of GST-fused di-W peptide were described previously . Briefly35S-Translabel (ICN Inc.) in Vpr-inducible medium and lysed in CHAPS buffer as previously described [HP16 co-transformants were radiolabeled with 150 μCi of escribed . Cell exescribed ,38. Brieescribed .X-J Y designed the experiments, constructed most Vpr mutants and wrote the manuscript. NR carried out the binding assays and tested the effect of Vpr mutants on yeast cells growth. JL selected and characterized the B4 GST-di-W-containing peptide. GD participated in the design of the study and critically evaluated the manuscript. EAC participated in the design of the study and coordinated it. All authors read and approved the final manuscript.


The guanine nucleotide exchange factor C3G (RapGEF1) along with its effector proteins participates in signaling pathways that regulate eukaryotic cell proliferation, adhesion, apoptosis and embryonic development. It activates Rap1, Rap2 and R-Ras members of the Ras family of GTPases. C3G is activated upon phosphorylation at tyrosine 504 and therefore, determining the localization of phosphorylated C3G would provide an insight into its site of action in the cellular context.C3G is phosphorylated in vivo on Y504 upon coexpression with Src or Hck, two members of the Src family tyrosine kinases. Here we have determined the subcellular localization of this protein using antibodies specific to C3G and Tyr 504 phosphorylated C3G (pY504 C3G). While exogenously expressed C3G was present mostly in the cytosol, pY504 C3G formed upon Hck or Src coexpression localized predominantly at the cell membrane and the Golgi complex. Tyrosine 504-phosphorylated C3G showed colocalization with Hck and Src. Treatment of Hck and C3G transfected cells with pervanadate showed an increase in the cytosolic staining of pY504 C3G suggesting that tyrosine phosphatases may be involved in dephosphorylating cytosolic phospho-C3G. Expression of Src family kinases or treatment of cells with pervanadate resulted in an increase in endogenous pY504 C3G, which was localized predominantly at the Golgi and the cell periphery. Endogenous pY504 C3G at the cell periphery colocalized with F-actin suggesting its presence at the subcortical actin cytoskeleton. Disruption of actin cytoskeleton by cytochalasin D abolished phospho-C3G staining at the periphery of the cell without affecting its Golgi localization.These findings show that tyrosine kinases involved in phosphorylation of C3G are responsible for regulation of its localization in a cellular context. We have demonstrated the localization of endogenous C3G modified by tyrosine phosphorylation to defined subcellular domains where it may be responsible for restricted activation of signaling pathways. Guanine nucleotide exchange factors (GNEFs) are components of signaling pathways that link transmembrane receptors to intracellular GTPase family members regulating a wide variety of cellular functions such as proliferation, differentiation, adhesion and apoptosis. C3G RapGEF1) is an ubiquitously expressed GNEF for Ras family proteins that particularly targets Rap1, Rap2 and R-Ras  is an ub. It has The C-terminus of C3G, which shows homology to CDC25, harbors the catalytic domain. The central region of C3G, which spans about 300 residues, has polyproline tracts with the ability to bind to SH3 domains of various proteins like Crk, p130 Cas, Grb2 and Hck ,18,27. NThe effectiveness and precision of intracellular signal transduction depends on protein-protein interactions that regulate enzyme activity as well as subcellular localization. Cell surface receptor activation leads to assembly of adaptor protein complexes at the plasma membrane, which serve to localize guanine nucleotide exchange proteins. Earlier, both endogenous as well as exogenously expressed C3G has been shown to localize to the cytoplasm and not to associate with plasma membrane ,30. SincWe have recently shown that Hck interacts with and phosphorylates C3G in vivo and we wished to determine if their interaction leads to changes in the subcellular distribution of C3G and whether pY504 C3G locates to specific subcellular domains. Cos-1 cells were transfected with C3G in the presence or absence of Hck and immunostained using anti C3G antibodies. As shown in Fig. To determine the subcellular distribution of phospho-C3G, which is known to be the activated form, specificity of a rabbit polyclonal phosphorylation site-specific antibody (pY504-C3G) was verified by examining its reactivity using cell lysates expressing C3G or Y504F-C3G alone or with Hck. As shown in Fig. We wished to determine whether C3G that colocalizes with Hck was the phosphorylated component and therefore used pY504-C3G antibodies to determine the localization of pY504 C3G in cells expressing Hck and C3G. As shown in Fig. In the above experiments phosphorylation and localization of C3G was studied using exogenously expressed protein and we wished to determine whether endogenous C3G could be phosphorylated and similarly targeted. Towards this end we checked the phosphorylation of endogenous C3G under conditions of Src and Hck overexpression or upon activation of cellular tyrosine kinases by pervanadate treatment. C3G protein is expressed as a doublet of about 140–150 kDa, which are products of two differentially spliced mRNAs . Whole cThe localization of endogenous pY504 C3G to the Golgi was examined in Cos-1 cells transfected with Hck and VSVG-GFP. Immunostaining for pY504 C3G was seen predominantly at the cell periphery and the Golgi Fig. , which wThe localization of pY504 C3G formed by the activation of endogenous tyrosine kinases was determined in cells treated with pervanadate, which is known to activate Src family kinases, in addition to inhibiting tyrosine phosphatases -37. PervIndirect immunoflourescence was performed to determine the localization of endogenous pY504 C3G formed by the activation of intracellular tyrosine kinases. Cos-1 and HeLa cells were stimulated by pervanadate and as seen in Fig. We have observed that pervanadate treatment increased tyrosine phosphorylation of endogenous C3G. Since overexpression of Src as well as Hck results in phosphorylation of C3G, it was of interest to determine whether pervanadate induced tyrosine phosphorylation of C3G was mediated by Src family kinases within the cell. Cells were treated with PP2, a specific SFK inhibitor prior to PV treatment . As showC3G is involved in a variety of signaling pathways and therefore its dynamic localization under normal and activated situations may be physiologically relevant. In this study we demonstrate the limited subcellular distribution of Y504 phosphorylated C3G, which is predominantly targeted to the Golgi apparatus and the subcortical actin cytoskeleton. This localization has been substantiated by colocalization with a Golgi marker protein and F-actin respectively.Rap1, the substrate of C3G has been localized to the Golgi, lysosomal vesicles and cortical actin cytoskeleton . But, ofWe observed more exogenously expressed pY504 C3G in the cytoplasmic compartment under conditions of inhibition of tyrosine phosphatases suggesting that pY504 C3G may be targeted by cytosolic tyrosine phosphatases. This regulation may help in restricting the activity of C3G to specific compartments. We observe very little endogenous pY504 C3G in the cytosol when HeLa or Cos-1 cells are treated with pervanadate, which not only inactivates tyrosine phosphatases, but also activates tyrosine kinases. It is possible that upon PV treatment endogenous C3G present in the cells is phosphorylated at the sites of location of the activated kinases. Pervanadate treatment has been shown to increase phosphotyrosine staining at the cell periphery indicating activation of kinases present in this subcellular domain .Recently c-Src and Jak2 have been implicated in the phosphorylation of C3G in response to growth hormone stimulation of NIH 3T3 cells because dominant negative mutants of these kinases inhibit C3G phosphorylation . It was Adhesion dependent Src activation leads to Rap-1 activation mediated by Crk and C3G . FibroblThe activity of guanine nucleotide exchange factor C3G is known to be regulated by tyrosine phosphorylation and membrane targeting. Using phospho-specific antibodies, we directly demonstrate that expression of Src family kinases or pervanadate treatment of cells induces phosphorylation of C3G on Y504. Unlike C3G, which is mostly cytosolic, pY504C3G locates to the Golgi and subcortical actin cytoskeleton. Demonstration of the localization of the active component of C3G to the Golgi and subcortical cytoskeleton provides evidence for a possible function for C3G at these cellular compartments.HeLa and Cos-1 cells were cultured in DMEM supplemented with 10% FCS. Transfections were performed on cells grown as a monolayer in either 35 mm dishes or glass coverslips using the cationic lipid DHDEAB as described . Briefly2O2, with 100 mM solution of sodium orthovanadate. It was added to the cells within 5 mins of preparation. Golgi disruption was performed by treating the cells with 5 μg/ml of nocadazole for 30 min prior to fixation. To disrupt actin cytoskeleton, cells were treated with 1 μg/ml cytochalasin D for 20 mins. PP2 was added to cells 2 hrs before pervanadate treatment at a concentration of 10 μM to inhibit Src family kinases.Cells were subjected to pervanadate treatment by the addition of a freshly prepared solution of pervanadate at 50 μM conc. for 10 min prior to harvesting. Pervanadate stock solution (50 mM) was prepared by mixing equal volumes of 100 mM solution of HFull length human C3G cloned in pcDNA3-FLAG was kindly provided by Dr S Tanaka. Y504F mutant of C3G in which tyrosine 504 is mutated to phenylalanine cloned in a His tagged expression vector was provided by Dr M Matsuda. The wild type rat p59 Hck cDNA was cloned in the pCI plasmid (Promega) and has been described earlier . ExpressWhole cell lysates were prepared by lysing cells directly in Laemli's sample buffer and subjected to SDS-polyacrylamide gel electrophoresis. After transfer onto nitrocellulose membranes, they were processed for western blotting using the required primary antibodies. Detection was based on either color development using alkaline phosphatase conjugated secondary antibodies or on chemiluminescence using horse radish peroxidase conjugated secondaries.Cells were processed for immunoflourescence staining as described earlier . The priGNEF – guanine nucleotide exchange factorSFK – Src family kinasePV – PervanadatepY504 C3G – Tyrosine 504 phosphorylated C3GVSVG – Vesicular stomatitis virus glycoproteinDMEM – Dulbecco's modified Eagle's mediumFITC – Fluorescein isothiocyanateVR designed and carried out the experiments, analysed the data and drafted the manuscript. GS helped with designing the experiments, analyzing the data and writing the manuscript. AR provided technical help for western blotting and indirect immunoflourescence experiments.


We sought to evaluate the hypothesis that the high incidence of cutaneous melanoma in white persons in central Alabama is associated with a predominance of Irish and Scots descent.Frequencies of country of ancestry reports were tabulated. The reports were also converted to scores that reflect proportional countries of ancestry in individuals. Using the scores, we computed aggregate country of ancestry indices as estimates of group ancestry composition. HLA-DRB1*04 allele frequencies and relationships to countries of ancestry were compared in probands and controls. Results were compared to those of European populations with HLA-DRB1*04 frequencies.Ninety evaluable adult white cutaneous melanoma probands and 324 adult white controls reported countries of ancestry of their grandparents. The respective frequencies of Ireland, and Scotland and "British Isles" reported countries of ancestry were significantly greater in probands than in controls. The respective frequencies of Wales, France, Italy and Poland were significantly greater in controls. 16.7% of melanoma probands and 23.8% of controls reported "Native American" ancestry; the corresponding "Native American" country of ancestry index was not significantly different in probands and controls. The frequency of HLA-DRB1*04 was significantly greater in probands, but was not significantly associated with individual or aggregate countries of ancestry. The frequency of DRB1*04 observed in Alabama was compared to DRB1*04 frequencies reported from England, Wales, Ireland, Orkney Island, France, Germany, and Australia.White adults with cutaneous melanoma in central Alabama have a predominance of Irish, Scots, and "British Isles" ancestry and HLA-DRB1*04 that likely contributes to their high incidence of cutaneous melanoma. The incidence of cutaneous melanoma in Alabama is high (16.9 and 8.6 per 100,000 men and women, respectively, in 1998) . LifestyIt has been hypothesized that the high incidence of melanoma in Alabama, a Sunbelt state with a mild climate, is due to the settlement of Alabama by a predominance of persons of Irish and Scots descent -10. In tThe performance of this work was approved by the Institutional Review Boards of the University of Alabama at Birmingham and Brookwood Medical Center. All subjects were adults (≥ 18 years of age) who were central Alabama residents; each identified himself/herself as white. Persons with melanoma were diagnosed in routine medical care in the interval 1981 – 2002, but were otherwise unselected. We excluded persons of sub-Saharan African or African American descent, because most of these persons have non-European ancestry and their incidence of cutaneous melanoma is much lower than in white persons in central Alabama [Cutaneous melanoma was diagnosed as previously described . HLA-DRBAdult control subjects residing in central Alabama were recruited in two groups. The first group consisted of 83 spouses of persons with cutaneous melanoma and randomly recruited subjects. The second group consisted of 260 unselected volunteers who completed the present questionnaire (described below); they were recruited from hospital workers, employees of two universities, spouses of patients who attended a hematology/medical oncology outpatient clinic, and members of the general public encountered in two retail shopping malls. In both groups, we excluded persons who were known to be relatives of other study participants. We did not evaluate medical histories or perform physical examinations in control subjects. These data were pooled to yield a group of 343 unrelated controls, of whom 24 were eliminated because they did not know the country of ancestry of any of their grandparents (as indicated below). This left a group of 319 control subjects whose data were deemed evaluable for the present study. HLA typing was performed on some control subjects as indicated below.A one-page questionnaire was designed to permit each study participant to indicate the countries of ancestry of his/her paternal and maternal grandparents. This method is identical to that previously validated in a study of the ancestry of hemochromatosis probands ; in partWe defined aggregate country categories as the composite of reports from respective countries . ReportsWe tabulated the number of participants who reported specific countries of ancestry or aggregate country categories (as defined above) for one or more grandparents. This method is identical to that previously described ; in partThe questionnaire and interview reports from each participant were evaluated to yield individual country of ancestry scores that reflect proportional national ancestry, as previously described . We alsoAn index of Celtic ancestry was computed in a manner similar to that used for country of ancestry indices. In the present analysis, persons who reported one or more grandparents whose country of origin was Ireland or Scotland were defined as "Celts." Other participants were defined as "non-Celts." We did not use grandparental surnames or maiden names to quantify the degree of Celtic ancestry as did previous investigators ,19.A manual and computerized literature search was performed to identify reports of HLA-DRB1*04 phenotype frequencies in white persons with or without melanoma in countries or areas of Europe and Australia that are known to have had Celt settlements The HLA-DRB1*04 phenotype frequency among white control subjects in central Alabama is approximately 0.2148, based on the combined data from two previous reports ,20. Thust test, as appropriate. However, Student t-test can not be used to compare country of ancestry data groups in which all values are 0 (no variability). Accordingly, we arbitrarily assigned a country of ancestry datum of 0.25 for one person in each proband country group for which there were no actual country of ancestry reports. Student t-test was then performed using this modified data group; estimated p values from these tests are displayed in parentheses. A p value < 0.05 was defined as significant. Odds ratios (OR) were calculated as described by Woolf [The dataset consisted of observations on 90 cutaneous melanoma probands and two groups of controls (83 interview subjects and 261 questionnaire responders, respectively). A computer spreadsheet (Excel 2000, Microsoft Corp., Redmond, WA) and a statistical program (GB-Stat, v. 8.0 2000, Dynamic Microsystems, Inc., Silver Spring, MD) were used to perform the present analyses. In a preliminary evaluation, we determined that the proportions of men and women, mean ages, country of ancestry reports, and frequencies of country of ancestry reports were not significantly different in the two control groups. Therefore, we pooled data from the two groups for comparison with those of melanoma probands. Frequencies of men and women, clinical abnormalities, and countries of ancestry were counted. General descriptive data are presented as percentages or mean ± 1 S.D. Comparisons between groups were tested for statistical differences using chi-square analysis, Fisher exact test or two-tailed Student by Woolf .There were reports from 90 evaluable probands (46 men, 44 women). Their mean age at the time of participation in the present study was 49 ± 16 years (range 19 – 80 years).There were reports from 319 evaluable control subjects (128 men, 191 women). The percentages of men and women in the control group were similar to those in the cutaneous melanoma probands . Their mean age at the time of participation in the present study was 43 ± 16 years (range 18 – 82 years). The mean age of the cutaneous melanoma probands was significantly greater than that of the control subjects .No person declined to participate in the study, and there were no incomplete, equivocal, or unintelligible questionnaire or interview reports. Some participants reported that they were unaware of their ancestry due to adoption, family estrangement, or disinterest in genealogy. Ninety of 110 cutaneous melanoma probands (81.8%) and 319 of 347 control subjects (91.3%) provided reports for at least one of four grandparents; these differences were significant (p = 0.0026). Data from these participants were included for further analysis. Thus, there were reports from 90 evaluable cutaneous melanoma probands and 319 evaluable control subjects. Among evaluable cutaneous melanoma probands, the mean number of countries reported was 2.2 ± 1.1 (range 1 – 5 countries). The mean number of countries reported by evaluable control subjects was similar (2.4 ± 1.1 countries (range 1 – 7 countries); p = 0.2246).These data are displayed in Table These data are displayed in Table HLA-DRB1 phenotypes were available for 63 of the 90 present cutaneous melanoma probands. We divided the probands for which we had both country of ancestry data and HLA-DRB1 phenotypes into "Celts" (n = 19) and "non-Celts" (n = 44), as defined above. There was no significant difference in the frequency of HLA-DRB1*04 in the two groups . Because this finding was unexpected and differed from that of previous reports  we then We identified HLA-DRB1*04 phenotype frequency estimates of > 0.2148 from England, Ireland, Scotland, Wales, Orkney Islands, Brest, Germany, and Australia, areas populated predominantly by persons of Celtic ancestry ,13,20,22The present results indicate that England, Ireland, Scotland and the aggregate "British Isles" are the countries of ancestry reported most frequently by cutaneous melanoma probands and control subjects in central Alabama. Germany is another country of ancestry often reported by the present study participants. These results are consistent with historical accounts of early migrations of persons of English, Irish and Scots descent into central Alabama -26, with"Native American" ancestry, especially Cherokee or Creek heritage, was reported by many of the present study participants, and this is consistent with accounts of early Alabama history ,30-32 anThe percentages of men and women were not significantly different in cutaneous melanoma probands and control subjects. Analyses of the two study groups indicate that age is not significantly correlated with country of ancestry indices. The mean age of cutaneous melanoma probands was significantly greater than that of control subjects. However, the mean number of countries reported by cutaneous melanoma probands and control subjects did not differ significantly. Thus it appears that diagnosis of cutaneous melanoma or greater age is not associated with greater interest or knowledge in personal ancestry, although this is unproven.30.0% of cutaneous melanoma probands and 30.1% of control subjects did not know the country of ancestry of any of their four grandparents and were thus declared inevaluable. Many others did not know the ancestry of some of their grandparents. Some participants reported that they were unaware of their ancestry due to adoption, family estrangement, or disinterest in genealogy. Other participants could have been incorrect in their reporting. The percentages of evaluable cutaneous melanoma probands and control subjects who reported grandparents in the "Don't Know" category were similar. Altogether, it is unlikely that exclusion of subjects who did not know the country of ancestry of each of their grandparents would significantly change the outcomes of the present study. The overall trends in frequency of country reporting and country of ancestry indices in "British Isles" and "Europe Not British Isles" categories in the present study were similar. This suggests that uncertainty of participants about the exact degree of country of ancestry of some of their grandparents was probably not a significant contributor to the major conclusions of the present study. It is possible that control subjects who were included also had cutaneous melanoma. Nonetheless, it is unlikely that identification of a presumably small number of undiscovered control subjects with cutaneous melanoma would significantly change the conclusions of the present study.Previous studies reported that Celtic ancestry is a risk factor for cutaneous melanoma, particularly when people of Celtic ancestry inhabit areas of high flux of ultraviolet radiation ,6. PersoThe present observations are consistent with those of another study in Alabama in which white persons with hemochromatosis, a disease thought to be of Celtic origin ,34, repoUsing an index of Celtic ancestry that included grandparental surnames, maiden names, and country of birth to evaluate persons in Wales, other investigators observed that "high-scoring Celts" were significantly more likely to have Fitzpatrick skin type I or II (poor ability to tan, and tendency to burn easily after sun exposure)  than nonObservations from previous studies demonstrate that the frequency of positivity for the HLA-DRB1*04 phenotype is significantly greater in Alabama cutaneous melanoma probands than in control subjects ,9,20. ThInvestigators in Texas and England reported that the HLA genotype DQB1*0301 influences either susceptibility to and/or severity of melanoma ,36-38. TThe highest frequencies of HLA-DRB1*04 in Europe occur in England, Wales, Ireland, the Orkney Islands, and the Brest area of France . These gOur observations and those of others suggest that certain HLA genotypes may be markers for Celtic ancestry ,14. It hThe present results support our hypothesis, and are also consistent with the association of cutaneous melanoma with Ireland, Scotland and "British Isles" ancestry, HLA-DR phenotypes, and estimations of a northern European somatic phenotype (eye color, hair color, amount of melanin in the skin) previously reported in white persons who reside in central Alabama ,4,8,9. TNone declared.RTA assessed the patients and participated in assessing the control population, conceived the study, participated in data collection, laboratory evaluation of the patients and controls, statistical evaluation, and wrote part of the manuscript. EHB participated in assessed the control population and in data collection. WWH participated in assessed the control population and in data collection. ALD participated in assessed the patient and control populations, in data collection and laboratory evaluation of the patients and controls. RCPG participated in conceiving the study. JCB participated in conceiving the study, assessing the control population, statistical evaluation and wrote part of the manuscript. All authors approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:


Simpson-Angus Scale (SAS) is an established instrument for neuroleptic-induced parkinsonism (NIP), but its statistical properties have been studied insufficiently. Some shortcomings concerning its content have been suggested as well. According to a recent report, the widely used SAS mean score cut-off value 0.3 of for NIP detection may be too low. Our aim was to evaluate SAS against DSM-IV diagnostic criteria for NIP and objective motor assessment (actometry).Ninety-nine chronic institutionalised schizophrenia patients were evaluated during the same interview by standardised actometric recording and SAS. The diagnosis of NIP was based on DSM-IV criteria. Internal consistency measured by Cronbach's α, convergence to actometry and the capacity for NIP case detection were assessed.Cronbach's α for the scale was 0.79. SAS discriminated between DSM-IV NIP and non-NIP patients. The actometric findings did not correlate with SAS. ROC-analysis yielded a good case detection power for SAS mean score. The optimal threshold value of SAS mean score was between 0.65 and 0.95, i.e. clearly higher than previously suggested threshold value.We conclude that SAS seems a reliable and valid instrument. The previously commonly used cut-off mean score of 0.3 has been too low resulting in low specificity, and we suggest a new cut-off value of 0.65, whereby specificity could be doubled without loosing sensitivity. Reported prevalences for neuroleptic-induced parkinsonism (NIP) in schizophrenia patients are usually in the range 19% to 36% . As NIP Simpson-Angus Scale (SAS) is a 10-item rating scale that has been used widely for assessment of NIP in both clinical practice and research settings . It consAccording to our recent study  there waAccelerometric methods have been developed to identify and monitor motor NIP symptoms, such as tremor ,19 and hThe discrepancy between SAS and DSM-IV based NIP prevalence estimates as well as other above mentioned shortcomings suggest that SAS needs an evaluation as a method to assess NIP severity and to find reliably NIP cases.Our aims were to check the internal consistency of SAS, improve the convergence between DSM IV and SAS based NIP case finding, and to evaluate how well the scale measures objective motor symptoms verified by actometry.We recruited 99 chronic schizophrenic institutionalized adult patients from a state nursing home in central Estonia . InclusiAn experienced clinician (SJ) assessed all the subjects to identify NIP cases in accordance with DSM-IV. The DSM-IV diagnostic criteria for other neuroleptic-induced movement disorders (NIMD) were also checked because of frequent comorbidity and common aetiology. Clinical NIP symptoms were assessed by SAS and the motor activity during rest was measured by actometry. Each item of the 10-item SAS is rated on a 5-point scale (0–4), and the mean score is obtained by adding the items and dividing by 10 . NeuroleThe actometric recording was performed during sitting in a standardized clinical interview for 30 minutes, a method described previously as measuring "controlled rest activity" ,21. ContCronbach's α was assessed to evaluate the internal consistency of the scale. The correlations between the lower limb activity (the mean of right and left ankle movement indices) and individual item scores and mean SAS scores were analysed. Differences between the NIP and non-NIP, as well as the NIMD and the non-NIMD groups in the SAS mean score and lower limb activity were analysed. The performance of SAS mean score and individual item scores in case identification was evaluated by receiver operating characteristics (ROC) analyses against DSM-IV NIP diagnosis. Validity coefficients  for different mean SAS score thresholds were calculated. To explore the discriminatory power of each single SAS item we performed ROC analyses for each item separately. We also explored the effect on the validity coefficients of merging the six rigidity items of SAS into one single item, to de-emphasise the influence of rigidity on the mean SAS score. The Spearman test was used to correlation analysis and the Mann-Whitney 2-tailed U-test for the comparison between two groups because of the non-normal distribution of the data. The software used in analyses was SPSS 11.0. .Of the 99 participants, 45 (45.5%) were male and 54 (54.5%) female. The mean age was 49.7 (SD 9.5) years. The mean continuous treatment in hospital or in nursing home was 13.6 (SD 9.0) years. Seventy-nine (79.8%) patients used conventional antipsychotics (70 on low-dose, and 9 on high-dose neuroleptics) and 20 (20.2%) used clozapine (one was receiving clozapine combined with sulpiride). Low-dose antipsychotics in this study were haloperidol, cyclopentixol, perphenazine and fluphenazine; high dose antipsychotics were chlorpromazine, thioridazine, levomepromazine, chlorprotixen and sulpiride. Sixteen (16.2%) patients were receiving combinations of typical antipsychotics (either predominantly low-dose [N = 10] or predominantly high-dose [N = 6] neuroleptic regimens), and 63 (63.6%) were receiving monotherapy . No new atypical antipsychotics were used. The mean daily chlorpromazine equivalent conditions. The prevalence of any NIMD according to DSM-IV was 61.6% in the whole sample. Cronbach's α for SAS was 0.79. dose  was 328 The SAS mean score for DSM-IV NIP patients (1.24, SD = 0.44) was significantly higher from that (0.56, SD = 0.33) of non-NIP patients (U = -6.90, p = 0.000). The mean scores of each single SAS item are presented in Table Actometric data was missing for one male patient due to non-co-operation. The median lower limb activity for NIP patients was not significantly higher than that of non-NIP patients (U = -0.46, p = 0.643). The median lower limb activity for NIMD patients was significantly higher from that of non-NIMD patients (U=-2.66, p = 0.008).The SAS mean score did not correlate significantly with actometric lower limb activity either in the whole population (r = 0.04, p = 0.717), in the NIP group (r = -0.29, p = 0.192), or in the pure NIP subgroup (r = -0.21, p = 0.587). Even after a post-hoc analysis of co-variance in the whole population, where the effect of akathisia  and tardive dyskinesia (AIMS severity score) were controlled for, no significant correlation between SAS mean score and the lower limb activity could be found (r = 0.07, p = 0.494).The tremor item of the SAS correlated significantly with the lower limb activity in the whole population (r = 0.25, p = 0.013) but not in the NIP population (r = 0.26, p = 0.248) or in the pure NIP subgroup (r = 0.51, p = 0.160). No correlation was evidenced between the hypokinesia item of the SAS and lower limb activity in the whole population (r = -0.07, p = 0.513) either in NIP population (r = -0.24, p = 0.290) or in pure NIP subgroup (r = -0.37, p = 0.797).No correlation was evidenced between the mean of rigidity items of the SAS and lower limb activity in the whole population (r = -0.12, p = 0.256) either in NIP population (r = -0.37, p = 0.090) or in pure NIP subgroup (r = -0.30, p = 0.426).ROC-curve for screening performance of SAS mean score is presented in Fig ROC-curve for screening performance of SAS mean with single averaged rigidity item was clearly inferior to the original SAS mean curve with AUC of 0.80 (CI = 0.70–0.89).The screening performances of the individual SAS items for NIP case finding are shown at Table As SAS elbow rigidity item had case finding power similar to SAS mean score, we calculated optimal cut-off for this item. Cut-off threshold of 1.5, with sensitivity of 0.826 and specificity of 0.974, was superior to cut-off threshold of 0.5 with sensitivity of 0.957 and specificity 0.553.Our study aimed to evaluate some of the characteristics of the SAS and its utility for identifying and measuring NIP in a naturalistic schizophrenia sample. The internal consistency of SAS was satisfactory, which suggests sufficient reliability for the scale. We compared the SAS with the DSM-IV to assess its discriminant validity and evaluate it in detecting NIP cases. The comparison with objective movement assessment aimed to estimate the concurrent validity of SAS in NIP severity measurement.As expected, the SAS had discriminant validity for a clinical diagnosis of NIMD. SAS mean score discriminated NIMD patients well from those without NIMD, and more specifically, also NIP patients from other patients. Actometry discriminated NIMD patients from non-NIMD patients, but did not identify DSM-IV NIP patients.According to ROC analysis the SAS had good case finding properties converging with the DSM IV NIP diagnosis. In our population the commonly used threshold 0.3 was inappropriate: according to our results the optimal cut-off point should be between 0.65 – 0.95 depending on the emphasis in the trade-off between sensitivity and specificity. We suggest that the new cut-off value for screening NIP could be 0.65, whereby specificity could be doubled without loosing any sensitivity. To be useful for diagnostic purposes a combination of high specificity and high positive predictive value (PPV) is reached at cut-off – 0.75 . To answUsing the single elbow rigidity item for case detection had the same (or slightly better) case detection capacity as the SAS mean score. This finding supports the use of elbow rigidity testing when assessing parkinsonism in clinical settings, as cut-off value 0.5 has good sensitivity and specificity for DSM-IV NIP.We found that SAS mean score did not correlate with actometric lower limb activity, and hypokinesia observed during gait item of SAS did not correlate with actometric motor activity during the 30-minute recording. There are a few explanations for that:First, actometry measures only the productive motor dimension of the parkinsonian symptoms while SAS takes into account also rigidity, gait, salivation and glabella tap, with a clear emphasis on rigidity. Lack of correlation with actometric findings in NIP subgroup indicates that tremor may not be the core feature of NIP. This is also supported by the small AUC for the tremor item of SAS.Secondly, we used lower-limb actometry while the clinical assessment by SAS and DSM-IV considered predominantly upper limbs. Parkinsonism may be more symptomatic in upper limbs, and the upper limb disturbances may have influenced our SAS and DSM IV assessments more than lower limb disturbances. Our findings indicate that lower limb actometry is not suitable for diagnosing NIP.Thirdly, diurnal naturalistic actometry may have more power in detecting hypokinesia.This study was limited to a few aspects of utility/validity of the SAS: internal consistency, convergence to DSM-IV NIP diagnosis and convergence to objectively measured motor activity. Many aspects of the scale's reliability (e.g. test-retest and inter-rater reliability) and validity (e.g. construct) were not evaluated.DSM-IV was used as a standard in this study, but there is not much data available on the validity of NIP criteria of the DSM-IV. A better golden standard in this study would probably have been an expert-consensus diagnosis. Furthermore, as there was only one rater for the scales, a cross-scale contamination issue might have occurred.It is known that with age the prevalence of spontaneous NIMD rises. Our material did not allow a thorough examination of the issue, but age correlated with SAS mean score in our sample.The measurement of motor activity here was purely quantitative; we did not assess the patterns of the disordered movements.As a conclusion, SAS seems be a reliable and a valid instrument. It performs well and similarly to DSM-IV in NIP case detection. The optimal SAS mean score cut-off value in a naturalistic population of neuroleptic-treated schizophrenia patients is higher than the commonly used 0.3. We suggest that the new cut-off value for screening NIP could be 0.65, whereby specificity could be doubled without loosing sensitivity. Combining SAS rigidity items does not seem to improve the performance of the scale.The author(s) declare that they have no competing interests.SJ contributed to the study design, collected the data, contributed to the analyses and data interpretation, made the literature search and was responsible for manuscript preparation with MMH.MMH contributed to study design, made most of the analyses and data interpretation, and was equally responsible for manuscript preparation with SJ.KT contributed to study design, statistical analysis, data interpretation and manuscript preparation.KW supervised the study design and contributed to statistical analysis, data interpretation and preparation of the manuscript.The pre-publication history for this paper can be accessed here:


There has been concern that the incidence of autism and other pervasive developmental disorders (PDDs) is increasing. Previous studies have been smaller, restricted to autism , included boys only, or have not been based on a national sample. We investigated time trends in the rates of diagnosis of pervasive developmental disorders.We analysed the rates of first diagnosis of pervasive developmental disorders among people registered with a practice contributing to the United Kingdom General Practice Research Database during the period 1988 to 2001. We included 1410 cases from over 14 million person-years of observation. The main outcome measures were rates of diagnosis of pervasive developmental disorders by year of diagnosis, year of birth, gender and geographical region.The rate increased progressively from 0.40/10,000 person-years (95% CI 0.30 to 0.54) in 1991 to 2.98/10,000 (95% CI 2.56 to 3.47) in 2001. A similar change occurred in the age standardised incidence ratios, from 35 (95% CI: 26–47) in 1991 to 365 (95% CI: 314–425) in 2001. The temporal increase was not limited to children born during specific years nor to children diagnosed in a specific time period. The rate of diagnosis of PDDs other than autism rose from zero for the period 1988–1992 to 1.06/10,000 person-years in 2001. The rate of diagnosis of autism also increased but to a lesser extent. There was marked geographical variation in rates, with standardised incidence ratios varying from 66 for Wales to 141 for the South East of England.Better ascertainment of diagnosis is likely to have contributed to the observed temporal increase in rates of diagnosis of PDD, but we cannot exclude a real increase. The term pervasive developmental disorder (PDD) refers to a range of disorders with onset in childhood characterised by abnormalities in the domains of language development, communication abilities and social interactions and by a rigid, repetitive pattern of behaviours and interests . Within There have been a number of reports that the incidence of PDD is increasing  and the As part of a case-control study to identify risk factors for autism and other PDDs , we haveThe GPRD was set up in 1987, then known as the VAMP  Research Bank . It consEach individual is assigned a unique identification number and all data that could identify individuals are removed from records before they are incorporated into the GPRD.st January 1988 to the 31st December 2001. Such children will have been eligible at some time in their life for MMR vaccination, an exposure of particular interest in the case-control study [The information analysed in the present study comprised the electronic medical records of children born in 1973 or later and registered with general practices contributing data to the GPRD during the period 1st January 1988 or the date at which the practice started contributing data to the GPRD. The end of the observation period was the date at which the practice stopped contributing data to the GPRD, or the 31st December 2001 if this was earlier. Individual patients were included in the study during the times within the observation period that they were registered with a practice contributing to the GPRD. The number of practices included in the GPRD varied during the study period, rising from 34 in 1988 to 557 in 1996 then falling to 380 by 2001.For a specific general practice, the start of the observation period for this study was taken as the later of either the 1Age- and gender-specific rates were calculated for people born in 1973 or later and registered with contributing general practices during each calendar year. Those registered for part of a particular year contributed time at risk for the period they were registered. People were classified by geographical location of their general practice by country for Wales, Northern Ireland and Scotland, and by the eight former administrative National Health Service regions for England.When a patient first registers with a practice contributing to the GPRD, past medical events and diagnoses judged to be clinically important by the general practitioner are recorded in the electronic record, with the date of each diagnosis where available. While a patient is registered with a contributing practice, all new diagnoses and all new drug therapies or changes in existing therapies are recorded contemporaneously. Cases were defined as people born in 1973 or later who had a first diagnosis of PDD entered into their general practice record while registered with a practice contributing to the GPRD during the study period. Cases were identified by searching the complete medical record of all people registered with participating practices during the study period for either OXMIS or READ clinical codes indicating a diagnosis of PDD: Appendix see . People On the basis of the recorded diagnoses patients were classified as 'autism' or 'other PDD'. Those with autistic disorders and similar presentations were grouped in the autism category and other descriptions (such as Asperger's syndrome) were grouped in the other PDD category.Crude rates and age standardised incidence ratios for first diagnosis of all PDDs, autism, and other PDDs were calculated by year of diagnosis and gender and for different geographical regions. Because of low numbers of cases in some years, indirect standardisation was used with rates during the whole study period as the standard rates. The age-standardised incidence ratios presented take into account variations in the age structure of the population in different time periods and different geographical regions. Rates of first diagnosis were calculated by year of diagnosis and year of birth, both in two year intervals.Ethical approval for the study was obtained from the Scientific and Ethical Advisory Group of the GPRD and from the ethics committee of the London School of Hygiene and Tropical Medicine.We identified 1410 persons with a first recorded diagnosis of PDD during the study period: 1097 were categorised as autism and 313 as other PDD, for 294 (94%) of whom the diagnosis was Asperger's syndrome (appendix).Table The person-years at risk varied from year to year because of changes in the number of contributing practices, which increased until the mid 1990s and then fell. During the study period incidence rates increased progressively for both males and females. The patterns of crude rates and of standardised incidence ratios were very similar. The overall rate increased seven-fold from 0.40/10,000 person-years (95% CI 0.30 to 0.54) in 1991 to 2.98/10,000 person-years (95% CI 2.56 to 3.47) in 2001. A similar change occurred in the age standardised incidence ratios, from 35 (95% CI: 26–47) in 1991 to 365 (95% CI: 314–425) in 2001. The overall male to female ratio was 4.8 and during 1991 to 2001 ranged from 2.7 to 8.4 (chi-squared test for heterogeneity, p = 0.21), with no clear temporal trend.Table Table There was a striking rise in the rate of diagnoses of other PDDs: from zero for the period 1988 to 1992 rising to 1.06/10,000 person-years by 2001. Over the study period the rate of diagnosis of autism also increased substantially. The patterns were broadly similar for males and females (data not shown). Few diagnoses of other PDDs were made before 1997, but by 2001 over one third of all diagnoses were PDDs other than autism.Table There was marked geographical variation in rates of first diagnosis, with the lowest rate in Wales being less than half the rate in the South East of England (chi-squared test for heterogeneity in SIRs by region, p < 0.001). In general, the regional variations were less marked for autism than for other PDDs and the ratio of the diagnoses of autism compared with other PDDs varied greatly between areas (chi-squared test for heterogeneity, p = 0.0006).There was around a 10-fold increase in the rate of first recorded diagnoses of PDDs in United Kingdom general practice medical records from 1988–92 to 2000–01 table . If thesSome of the general practices contributing data to the GPRD will provide anonymised copies of hospital letters and specialist reports on individual patients. Of patients with a recorded diagnosis of PDD in the GPRD, including prevalent cases when first registered, 446 were registered with 203 general practices willing to provide this service. For 80 of these, medical records were not available as the patient was no longer registered with the general practitioner. We obtained complete case records including copies of hospital clinic letters and specialist reports for 318 (87%) of the remaining 366 people. These were reviewed by a psychologist (LH) and a random sample of 50 records were also reviewed by a child psychiatrist (EF), both of whom have long experience in the field of autism. They judged that a PDD was likely to be present in 294 children (92.5%). For theOf the 211 cases reviewed and included in this paper, 5 (2.4%) were classified as 'other-PDD' on the basis of their electronic record only, whereas in the validation, 32 (15.1%) met diagnostic criteria for a PDD other than autism. Thus it is likely that a proportion of people in the 'autism' diagnostic category in this paper have a form of PDD other than autism. The inaccuracy of diagnostic descriptions of different PDDs within the GPRD is likely to reflect changes in the definition of PDD over the past two decades, in particular a broadening of PDDs other than autism ,17. ManyIn the late 1980s and early 1990s autism was only likely to be diagnosed by specialist child psychiatrists who increased in number by 40% between 1988 and 2001 . Through the 1990s developmental and community paediatricians began to diagnose PDDs, and this, combined with increased awareness of autism and PDDs among the general public, may have contributed to increased ascertainment of the disorders [It is likely that children with PDDs other than autism will generally be first diagnosed at later ages than children with autism. Greater ascertainment of other PDDs during the latter part of the study period table  could haBecause of low numbers of cases in some years, indirect standardisation was used to calculate SIRs. When comparing SIRs, marked differences in the age distribution of the populations being compared can lead to a biased comparison . HoweverThe accuracy of the denominator data may have changed during the study period. When patients move geographical area they may delay registering with a new general practice until they have a specific reason to visit them. Thus our estimates of person-years at risk may be too low for those moving into a practice and too high for those moving out. Person-years at risk may also be inflated as patients who emigrate may omit to inform their general practitioner and, furthermore, administrative delays and errors may result in individuals being registered with more than one general practitioner. For example, in 1997 in England 51 million people were registered with a general practitioner  whereas A previous study based on the GPRD assessed time trends in the diagnosis of autism from 1988 to 1999 ,25. The This is one of the largest studies undertaken of trends in the incidence of autism and other PDDs. We describe striking increases in the rates of diagnosis of these conditions. However, much of the increase may be due to better ascertainment related to changes in diagnostic practice and improved recognition of the conditions. While review of clinical records confirmed that over 90% of diagnoses of PDDs recorded by general practitioners were likely to be correct, the nature of the study precluded us from assessing how often children with PDDs were not diagnosed. Thus the extent to which the increases in incidence we document are true increases in disease is uncertain.PDD pervasive developmental disorderGPRD General Practice Research DatabaseThe authors declare that they have no competing interests.AH, LS, EF, LR and PS designed the study. LH and EF undertook the validation of case reports. CC analysed the data. LS drafted the paper. All authors commented on drafts and approved the final version of the paper.The pre-publication history for this paper can be accessed here:Appendix: Codes used to identify cases, numbers identified, and diagnostic classification used in this paperClick here for file


Even though the catecholamines (dopamine, norepinephrine and epinephrine) have been detected in plants their role is poorly documented. Correlations between norepinephrine, soluble sugars and starch concentration have been recently reported for potato plants over-expressing tyrosine decarboxylase, the enzyme mediating the first step of catecholamine synthesis. More recently norepinephrine level was shown to significantly increase after osmotic stress, abscisic acid treatment and wounding. Therefore, it is possible that catecholamines might play a role in plant stress responses by modulating primary carbon metabolism, possibly by a mechanism similar to that in animal cells. Since to date no catecholamine receptor has been identified in plants we transformed potato plants with a cDNA encoding human dopamine receptor (HD1).Tuber analysis of transgenic plants revealed changes in the activities of key enzymes mediating sucrose to starch conversion (ADP-glucose phosphorylase and sucrose synthase) and sucrose synthesis (sucrose phosphate synthase) leading to altered content of both soluble sugars and starch. Surprisingly the catecholamine level measured in transgenic plants was significantly increased; the reason for this is as yet unknown. However the presence of the receptor affected a broader range of enzyme activities than those affected by the massive accumulation of norepinephrine reported for plants over-expressing tyrosine decarboxylase. Therefore, it is suggested that the presence of the exogenous receptor activates catecholamine cAMP signalling in plants.via cAMP signalling pathway.Our data support the possible involvement of catecholamines in regulating plant carbon metabolism  The catecholamines (dopamine, norepinephrine and epinephrine) are a group of biogenic amines possessing a substituted 3, 4-dihydroxy phenyl ring that are widespread in the animal kingdom; but they have also been detected in plants ,2. The rSolanum tuberosum plants transformed with pHD1-BinAR, a plasmid carrying a cDNA for the human dopamine receptor under the control of the CaMV 35S promoter  was recently investigated. A sensitive GCMS method based on the analysis of the trimethylsilylated catecholamine derivatives was developed to monitor the presence and concentration of these compounds and related metabolites. Based on the retention times and the mass spectra of standards the presence of dopamine, norepinephrine and a new compound normetanephrine in potato leaves and tubers was clearly detected .In contrast to the previous studies performed on plants over-expressing tyrosine decarboxylase , which cFollowing our recent finding that the action of dopamine and norepinephrine in potato is on starch mobilization, we decided to analyze transgenic tubers expressing dopamine receptor for soluble sugars and starch content. All transgenic plants showed decreased starch content, with levels that ranged from 20 to 60% percent of the wild type. This was accompanied by a significant increase in soluble sugar concentration ranging from 2.7 to 1.15 fold in comparison to Desiree Figure . ConcentChanges in carbohydrate are most likely responsible for the altered phenotype of transgenic HD1 tubers. Reduced tuber mass can be explained by decreased starch content whereas increased tuber number by the increase of soluble sugar concentration.Under normal growth conditions the major flux in potato tuber carbon metabolism is the conversion of sucrose through hexose phosphates to starch .Since HD1 plants were characterized by changed concentrations of both soluble sugars and starch we measured the activities of enzymes involved in this pathway. Sucrose transported from leaves is symplastically unloaded from the phloem and degraded by sucrose synthase (SuSy). ADP-glucose phosphorylase (AGPase) converts glucose-1-phosphate (Glu-1-P) into ADP-glucose, an immediate precursor of starch. Both SuSy and AGPase are considered as key enzymes for starch synthesis .Activities of AGPase and SuSy were significantly decreased in HD1 plants to 56% and 68% of the wild type level, respectively Figure . In agrePhosphoglucomutase (PGM) catalyzes the conversion of glucose-1-phosphate to glucose-6-phosphate. Tubers are characterized by the presence of cytosolic and plastidial isoforms of phosphoglucomutase. Repression of either of them results in plants with decreased starch levels pointing out the importance of the enzyme for starch accumulation ,19. The To establish whether enhanced starch mobilization also contributed to the observed decreases in starch content we measured the activity of starch phosphorylase. In two out of the four examined transgenic lines the activity of starch phosphorylase was significantly increased, further contributing to decreased starch content of HD1 plants Figure Moreover HD1 expression led to activation of sucrose phosphate synthase (SPS), responsible for sucrose production. Maximum SPS activity (measured wih saturating substrates, Vmax) only changed in two lines, whilst activity of the enzyme measured in the assay that contained limiting substrate concentration (Vmax/Km) and as a consequence 1/Km increased in all the lines.1/Km, correlated well with the sucrose content of transgenic tubers (cor -0.81) Figure .via the TCA cycle we measured the content of TCA intermediates. In all transgenic lines citric acid, isocitric acid and malate were significantly reduced, while fumarate showed a significant increase (Table The high concentrations of glucose and glc-6-P measured in the HD1 plants indicated changes in the glycolytic pathway. However, activities of glycolytic enzymes (hexokinase, phosphofructokinase and enolase) were not changed. The only exception was pyruvate kinase, which showed a significant decrease of activity in all transgenic lines Figure . To invese Table .In contrast to the vast knowledge concerning the role and action of catecholamines in mammals, very little is known about the physiological significance of catecholamines in plants. Since most of the components of animal catecholamine signaling pathway have been also identified in plants (G-proteins, cAMP, PKA homologs) the involvement of catecholamines in plant signalling pathways is possible. Recently, the analysis of transgenic plants over-expressing tyrosine decarboxylase, which accumulate a high quantity of catecholamines, suggested a possible signalling effect on plant primary metabolism. The increase of catecholamines resulted in decreased starch concentration but increased soluble sugars .The only component of mammalian catecholamine signaling pathway that to date has not been identified in plants is the catecholamine receptor. We transformed potato plants with a cDNA encoding human dopamine receptor (HD1) in order to analyze whether the presence of a receptor affects the endogenous catecholamine action. Western blot analysis showed that the protein was produced in transgenic plants and biochemical analysis of transgenic tubers revealed vast changes in carbohydrate metabolism and carbohydrate content. Surprisingly the catecholamine level was changed as well. It has to be pointed out that in contrast to plants over-expressing tyrosine decarboxylase, those expressing human dopamine receptor are characterized by increases of all known tuber catecholamines (dopamine, norepinephrine and normetanephrine). Whereas norepinephrine content was positively correlated with soluble sugars and negatively with starch, normetanephrine was considered as the product of norepinephrine turnover. Increased catecholamine content was accompanied by an increase of their precursors, tyramine and L-DOPA, suggesting upregulation of the biosynthetic pathway, mediated by tyrosine decarboxylase and tyrosine hydroxylase, respectively. It is hard to explain how expression of a human receptor triggers a positive loop leading to enhanced catecholamine synthesis and turnover. It is interesting to compare data on tuber carbohydrate levels from plants over-expressing tyrosine decarboxylase (TD) with those expressing human dopamine receptor. In both cases starch content is strongly decreased, this decrease was larger for HD1 plants (from 40% to 80%) than for TD tubers (from 12% to 60%) although the norepinephrine content was higher in TD plants  to the observed changes. Increased content of hexose-6-phosphates demonstrates that a direct inhibition of AGPase, rather than a substrate shortage may cause inhibition of starch synthesis. Alternatively the increased hexose phosphate levels may be due to increased starch degradation in response to elevated catecholamine levels. This is supported by the increased activity of starch phosphorylase in two of four transgenic lines. The inhibition of starch synthesis and accumulation of hexose phosphates was accompanied by an increase of sucrose synthesis. Two factors should be taken into consideration. First, that SPS is subject to allosteric activation by Glc-6-P and inhibition by Pi. Second, elevated catecholamine content led to a decrease of SPS Km suggesting increase of the enzyme catalytic activty. Sucrose phosphate synthase has many potential sites of phosphorylation and three of them were shown to influence its catalytic activity. In spinach leaf, phosphorylation of Ser 158 is responsible for enzyme downregulation in darkness, phosphorylation of Ser 229 enables binding of 14-3-3 proteins and down- regulates the enzyme whereas phosphorylation of Ser 424 under stress conditions stimulates SPS activity. There is a growing body of correlative evidence that the potato tuber SPS is regulated in an analogous manner to the leaf enzyme .Since the level of 14-3-3 proteins was not changed in any of the transgenic lines (data not shown) it is thus suggested that enzyme phosphorylation targeted to the stress site is responsible for its activity enhancement in HD1 plants.In mammals the action of epinephrine and norepinephrine is mediated by phosphorylation of enzymes involved in glycogen mobilization and synthesis. Very recent studies reported direct evidence that enzymes of starch metabolism (amylopectin synthesis) are regulated by protein phosphorylation and indicate a wider role for protein phosphorylation in the control of starch anabolism and catabolism . TherefoIn mammalian systems, catecholamines serve as stress hormones increasing as a result of stress. In order to see whether or not a similar response occurs in plants, leaves of potato plants were wounded and catecholamines levels prior to and 5, 10 and 13 min after wounding were determined. Although the data varied, there was a consistent increasing trend in concentration of dopamine, norepinephrine and normetanephrine . Very reIntroducing humane dopamine receptor into plant cells can be considered as controversial but the obtained data would argue for the value of our approach.Vast changes in the activities of key enzymes mediating carbon metabolism of potato tuber (in HD plants) led to a dramatic reduction of starch but increased sucrose content. The relation between catecholamine, primary carbon metabolism and stress seems possible. We speculate that similarly to situation in animal cells expression of HD1 in potato resulted in activation of the cAMP mediated signalling pathway. This can be supported by the result obtained for potato plants expressing another isoform of human dopamine receptor, HD2. In contrast to HD1, HD2 receptor does not affect activity of adenylate cyclase in animal cells.Similarly plants expressing HD2 showed no changes in carbohydrate metabolism (data not shown). The obvious next step would be further investigation of our plants with respect to their kinase activity as well as cAMP levels. In parallel we have made efforts to identify a plant dopamine receptor.Solanum tuberosum L. cv. Desiree) obtained from "Saatzucht Fritz Lange KG" (Bad Schwartau, Germany) were cultivated in a greenhouse in soil under 16 h light (22°C) and 8 h dark (15°C) regime. Plants were grown in individual pots and watered daily. For analysis, the leaves were harvested at noon from 30-day-old greenhouse grown plants and the tubers were harvested in September, 3 months after the transfer of the tissue culture plants to the greenhouse.Potato plants  were transformed with A. tumefaciens by immersing leaf explants in bacterial suspension. A. tumefaciens inoculated leaf explants were subsequently transferred to callus induction medium and shoot regeneration medium. Transgenic plants were pre-selected by using PCR with the primers for the respective phosphotransferase (kanamycin resistance) gene and then selected by northern blot analysis with a HD1 specific cDNA fragment as probe.The 1.3 kb SmaI, XbaI cDNA encoding HD1 from Homo sapiens ; [EMB: XX55760])), was ligated in the sense orientation into the same restriction site of the plant binary vector under the control of the 35S CaMV promoter and Nos terminator. The vector was introduced into the Total RNA was prepared from frozen plant material using the guanidinium hydrochloride method. Following electrophoresis (1.5% (w/v) agarose, 15% formaldehyde (w/v)), RNA was transferred to nylon membranes (Hybond N, Amersham, UK). Membranes were hybridised overnight at 42°C in 250 mmol sodium phosphate buffer (pH 7.2) containing 7% (w/v) SDS, 1% (w/v) bovine serum albumin (BSA) and 1 mM EDTA. Radioactively labelled full-length cDNA was used as a hybridisation probe. Filters were washed three times in 1 × SSC containing 0.5% (w/v) SDS at 65°C (highly stringent condition) or in the same buffer but at 42°C (medium stringent condition) for 30 min.2, 1 mM EDTA, 1 mM EGTA, 20%glycerol (v/v), 0.5 mM PMSF, 70 mM beta-mercaptoethanol) supplemented either with 0.1% or 1% TritonX- 100 (v/v). The assessment of the expression of HD1 gene by means of western blot analysis using rabbit IgG anti HD1 protein was conducted as described previously. Briefly, solubilised protein was run on 12% SDS polyacrylamide gels (w/v) and blotted electrophoretically onto nitrocellulose membranes (Schleicher and Schuell). Following transfer, the membrane was sequentially incubated with blocking buffer (5% (w/v) dry milk), and then with antibody directed against the HD1 protein (1:2000 dilution). Formation and detection of immune complexes were performed as previously described [Proteins were extracted from frozen plant material using extraction buffer E , heated for 15 min at 70°C, and centrifuged (5 min., 12000 g). Samples were diluted with water to 50% methanol concentration and extracted with chloroform (1:1 v/v). A portion of the water phase was dried under vacuum and used for derivatisation [-1 fresh weight).Frozen plant tissue (400 mg) was powdered in liquid nitrogen and extracted with methanol (4 ml per g5 min., 1000 g. Sa-1) at 37°C for 90 min and then acidic protons were derivatised with N-Methyl- N-trimethylsilyltrifluoroacetamide (MSTFA) at 37°C for 90 min. 2 μl of sample was used for analysis [-1. The temperature program was 5 min isothermal heating at 70°C, followed by a 5°C min-1 oven temperature ramp to 310°C and final 1 min heating at 310°C. The system was then temperature equilibrated for 6 min at 70°C prior to injection of the next sample. Mass spectra were recorded at 2 scan s-1 with an m/z 50 – 600 scanning range. The chromatograms and mass spectra were evaluated using the MSD ChemStation program (Hewlett Packard, Germany). As standards, dopamine, norepinephrine, normetanephrine, L-Dopa, tyramine and tyrosine (Sigma) were used. The recovery samples were spiked with dopamine, norepinephrine, normetanephrine, L-Dopa, tyramine and tyrosine; the estimated recoveries were 80, 93, 85, 95, 82, 90%, respectively. The following ions were used for quantification: ribitol 307; 319, L-Dopa 218; 267; 368, dopamine 174; 338; 426, norepinephrine 174; 355, 514, normetanephrine 174; 297; 456, tyramine 174; 264; 338, tyrosine 218; 280, 354. The amounts of catecholamines were determined from the ratio of peak areas of catecholamines to peak area of the internal standard (ribitol).The dried extracts were incubated in pyridine/methoxyamine 20 mg mL0 mg mL-12. 0.5 g ± 0.1 g tissue was homogenised in a chilled mortar in 2 ml of an extraction buffer containing 30 mM HEPES-NaOH, pH 6.9, 10 mM DTT, 1 mM MgSO4, 0.5 mM EDTA, 0.5% (w/v) BSA and 0.5% (w/v) PVP at 4°C. The homogenate was centrifuged at 16000 g for 10 min and the supernatant desalted using Sephadex G-25 columns. Enzyme activities were determined using the following published methods; SPS [Tissue was harvested, weighed and immediately frozen in liquid Nods; SPS ; SuSy, Pods; SPS ; PGM [33ods; SPS ; PGI, enods; SPS ; UGPase ods; SPS ; hexokinods; SPS  and starods; SPS .The t-tests were performed using the algorithm embedded into Microsoft Excel. The term significant is used when P < 0.05.AS carried out the metabolic analysis of transgenic plants and drafted the manuscript. AŚ carried out the construction and selection of transgenic plants and performed the statistical analysis. JS conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


Sustainable DNA resources and reliable high-throughput genotyping methods are required for large-scale, long-term genetic association studies. In the genetic dissection of common disease it is now recognised that thousands of samples and hundreds of thousands of markers, mostly single nucleotide polymorphisms (SNPs), will have to be analysed. In order to achieve these aims, both an ability to boost quantities of archived DNA and to genotype at low costs are highly desirable. We have investigated Φ29 polymerase Multiple Displacement Amplification (MDA)-generated DNA product (MDA product), in combination with highly multiplexed BeadArray™ genotyping technology. As part of a large-scale BeadArray genotyping experiment we made a direct comparison of genotyping data generated from MDA product with that from genomic DNA (gDNA) templates.Eighty-six MDA product and the corresponding 86 gDNA samples were genotyped at 345 SNPs and a concordance rate of 98.8% was achieved. The BeadArray sample exclusion rate, blind to sample type, was 10.5% for MDA product compared to 5.8% for gDNA.We conclude that the BeadArray technology successfully produces high quality genotyping data from MDA product. The combination of these technologies improves the feasibility and efficiency of mapping common disease susceptibility genes despite limited stocks of gDNA samples. In order to locate the disease variants involved in complex common disease it is now generally accepted that very large sample numbers will be required -4. Not oAt present, the gold standard method for generating gDNA from whole blood samples is through the process of immortalisation by transformation of the peripheral blood lymphocytes with Epstein-Barr Virus (EBV) . AlthougMolecular Staging, Inc. (MSI) (New Haven, CT, USA) have developed a method for whole genome amplification by Φ29 polymerase Multiple Displacement Amplification (MDA). It has been reported by the company that this method can reliably amplify the whole genome from gDNA, whole blood and other clinical samples -8. Each Recent studies have been conducted using MDA product from Amersham ,10, and ® (Assays-by-DesignSM, Applied Biosystems, Foster City, CA, USA) and Invader® (Third Wave Technologies, Inc., Madison, WI, USA) with gDNA.The BeadArray genotyping platform of Illumina™ Inc. (San Diego, CA, USA) offers a high-throughput, highly multiplexed and highly automated genotyping service facility . The BeaHLA-DRB1 genotype of each MDA sample was entirely concordant with the corresponding gDNA template, verifying the identity of each MDA sample and ruling out the possibility of contamination.We selected and sent to MSI 20 ng of 88 gDNA samples for amplification, from which an average of 200 μg of MDA product was yielded in 100 μl reactions. The yields ranged from 85 μg to 280 μg, with 61% of samples yielding between 100 μg and 200 μg. The When 100 ng of 448 gDNA samples were amplified using reagents supplied by MSI in kit form and the amplification carried out in-house, an average of 155 μg of MDA product was yielded in 150 μl reactions. The yields ranged from 31 μg to 260 μg, with 80% of samples yielding between 100 μg and 200 μg.INS) -23HphI (rs689), an allelic bias was observed in the MDA process, which resulted in the merging of the heterozygote cluster with the homozygote cluster of the major allele, making the correct assignments of genotype impossible, shown in Figure 2 [see INS in PCR reactions designed for the Pyrosequencing method  [INS may be situated in a sequence region that is predisposed to such allelic bias and the INS variable number of tandem repeats polymorphism, only 580 bp 5' to the -23HphI SNP, is a candidate for such an effect.Of 88 MDA products and their corresponding gDNAs tested at 95 SNPs using the TaqMan method of genotyping there were no samples that consistently failed to produce any data. This confirmed that, for all samples, amplification had been sufficiently successful for the TaqMan chemistry to perform at most SNPs. Genotype concordance rates between MDA product and gDNA and genotype failure rates are given in Table re 2 see . MDA prore 2 see , indicat Sweden) . These rFor 13 additional SNPs for which Invader genotyping was performed, comparison of genotypes generated from MDA product with those from gDNA are shown in Table Two SNPs were genotyped by TaqMan on the 448 samples amplified using reagents supplied by MSI in kit form (and the amplification carried out in-house), and on the corresponding gDNAs. The gDNA samples used for amplification had been extracted from whole blood. Genotype concordance rates between MDA product and gDNA and genotype failure rates are given in Table in silico assay design. These SNPs were selected for their relevance to a range of ongoing projects in our laboratory, located at genes of strong functional candidacy and within regions of linkage to type 1 diabetes e.g. the putative IDDM10 locus on chromosome 10p14-11. All SNPs were validated, having been identified either from empirically confirmed SNPs in dbSNP or from our own re-sequencing efforts. Based on ranking from the in silico design criteria [We commissioned Illumina to conduct a large-scale project using BeadArray genotyping technology involving 3,036 samples (2,950 gDNA samples and 86 MDA products) and 384 SNPs i.e. >1.1 million genotypes. In the first instance, 757 SNP sequences were sent to Illumina for criteria , 404 SNPAs well as excluding SNPs that fail to produce robust genotypes, the Illumina protocol excludes samples that do not consistently perform. Of the total number of samples 10.5% were excluded and as a consequence very few data points were missing from the data set, resulting in an apparently low genotype failure rate Table . Within As our samples were family-based, a quality control check of misinheritance rates was possible using PedCheck . Of the Within the panel of 384 SNPs attempted by Illumina, 17 were controls for which we had already produced genotyping data by either TaqMan or Invader methods, enabling an evaluation of the BeadArray data for concordance. Two of these 17 control SNPs failed to be converted to a working assay, giving 15 SNPs and a maximum of 2,503 samples that were genotyped in common. Excluding failed duplicates noted above, comparison of BeadArray genotypes with existing data revealed a concordance rate of 99.6% (129 discordant in 34,219 comparisons), indicating the compatibility of the non-excluded gDNA samples with BeadArray, and the quality of existing data. Of the 15 control SNPs, 11 had been genotyped using TaqMan and four using Invader. The concordance rates for each platform were 99.7% using TaqMan (104 discordant in 25,203 comparisons) and 99.6% using Invader (25 discordant in 9,016 comparisons) when compared with BeadArray data, showing no significant difference between the two platforms.t-test P-value >0.05 for every SNP).Within the BeadArray experiment described above were 86 MDA products and their corresponding gDNA samples. These data were directly compared for sample failure rate and genotype failure rate as shown in Table In this study we have evaluated the Φ29 polymerase MDA whole genome amplification method from MSI by assessing the compatibility of its product with the established TaqMan and Invader genotyping chemistries and with the highly multiplexed BeadArray genotyping platform. We have also evaluated Illumina's BeadArray genotyping platform for a large-scale experiment using gDNA.n = 88 in order to be confirmed. This result is comparable to the smaller study by Tranah et al. [et al. with fluorescent minisequencing on Amersham MDA products compared to gDNA [INS -23HphI, rs689).At 95 SNPs, comparison of TaqMan genotypes generated from MDA product and gDNA templates revealed a very good concordance rate but a higher failure rate for MDA product compared to gDNA. This would need be estimated in a sample size larger than the current  to gDNA . One maret al. [et al. [et al. [Compared to the yields indicated in Dean et al. , our ave [et al. . Further [et al.  protocolet al. using 2,320 SNPs and five samples [In the evaluation of MDA product in conjunction with BeadArray technology, the high concordance rate between genotypes obtained from MDA product and gDNA templates is encouraging. A concordance rate of 99.86% has been reported by Barker  samples . However samples . As withThe performance of MDA product is continuously being monitored in our laboratory. In a study blinded to all genotypers and database administrators, 288 family-based gDNA samples , were replaced with MDA product and left in continual use in our genotyping pipeline for 12 months. The change went undetected by all users. The failure rate for MDA was 3.34% for 15,921 genotypes, compared to 2.39% for 19,272 gDNA genotypes. Therefore, this improvement in the MDA performance for TaqMan is likely to be applicable to BeadArray, which improves the feasibility of mapping susceptibility loci in complex traits.When using a highly multiplexed, highly automated genotyping platform, slight reductions in the quality of template material are likely to have a greater adverse effect on data than in scenarios in which markers are assessed individually and manual scoring is undertaken. Our results indicate that MDA is an adequate solution for the vast majority of SNP markers, even in this highly multiplexed allelic assay platform.It is noted that 5.8% of markers that passed the Illumina acceptable scoring threshold were in fact showing high misinheritance rates in our family samples. This problem was at the same magnitude as TaqMan for individually genotyped markers. This highlights the importance of checking potential positive results with a second genotyping technology.MDA should allow the continuation of genetic analysis on archived DNA in researchers' freezers worldwide, providing the very necessary increases in sample sizes so urgently required ,2,17.The combination of BeadArray high throughput, multiplex genotyping and amplified DNA (MDA product) successfully produced high quality genotype data thereby improving the feasibility and efficiency of mapping common disease susceptibility genes despite limited stocks of gDNA samples.For both the validation experiments (MDA product as a template for TaqMan and Invader genotyping) and for the combined experiment (MDA product as a template for BeadArray genotyping) the same MDA samples were tested. We sent to MSI 20 ng (5 μl at 4 ng/μl) of 88 gDNA samples for amplification, which was performed as a service according to the protocol for human gDNA with the omission of the denaturing step . These gHLA-DRB1 and comparison made with data generated from the corresponding gDNA. HLA-DRB1 genotyping was performed using the Dynal Auto RELI™ SSO HLA-DRB Test system  for each gDNA sample and their MDA products. Although, these samples were amplified by MSI as a service, the reagents are also available from MSI in a kit form for amplification in-house. Following the amplification by MSI we have amplified 448 DNA samples by using the reagents in kit form and 100 ng (25 μl at 4 ng/μl) gDNA template in 100 μl reactions. In the interim, one major change to the MDA protocol had taken place, the inclusion of a denaturation of the DNA template prior to amplification. Previously no denaturation step took place. Two TaqMan markers were tested on these 448 samples and the genotype failure rate calculated.In order to verify the identity of each MDA-produced sample, genotyping was performed at SNP TaqMan assays were carried out for allelic discrimination, 8 ng of DNA (2 μl at 4 ng/μl) used in a 5 μl total reaction volume. TaqMan genotypes from the 88 MDA samples, described above, were compared with TaqMan genotypes generated from their corresponding gDNAs, at 95 SNPs, with a broad range of allele frequencies. The Invader method was used to genotype 13 additional SNPs on the same samples. Comparison was made between data generated from both templates by the measurement of genotype failure and genotype concordance rates.Of the 384 SNPs selected for genotyping 3,036 samples, 17 were control SNPs for which we had existing genotype data, generated by either TaqMan or Invader methods, with which comparison of genotype failure and genotype concordance rates were made. These 384 SNPs covered a broad range of allele frequencies.Incorporated into this experiment was an assessment of suitability and compatibility of the BeadArray genotyping method with MDA product. This involved 86 of the 88 amplified samples, described above, for which genotyping was attempted at all 384 SNPS. Concordance between genotypes generated from MDA product and gDNA templates, together with the genotype and sample failure rates of each template type were measured.In our laboratory we store genotyping data in a MySQL database on a Sun server. The volume of data expected from Illumina was the equivalent of 6 months' in-house genotyping. We separated phenotypic and pedigree information, which is associated with a sample, from genotype data, which is associated with a DNA plate and well position, with a link table to relate the two. Sample aliases are also supported, so that no recoding of identifiers is required, either to export or import Illumina data .RP participated in the design of the study, performed in-house genotyping experiments, and assisted in preparing the manuscript. HER prepared DNA and MDA amplified samples. BJB participated in the design of the study and assisted in the preparation of the manuscript. SN prepared DNA and MDA amplified samples. DS performed in-house genotyping experiments. MS prepared DNA and MDA amplified samples. RCJT participated in the design of the study and assisted in the preparation of the manuscript. AS participated in the design of the study. ACL and LJS performed bioinformatics. NMW managed the data and participated in its interpretation. JAT participated in the design of the study and assisted in the preparation of the manuscript. All authors read and approved the final version of the manuscript.INS -23 HphI SNP. Both the MDA product plots could not be scored. All plots represent the same individual samples with gDNA plots containing 8 additional samples.Figure 2 TaqMan and Invader fluorescence data plotted for the Click here for file