Computational and experimental evidence is given for alternative splicing at the unusual GYNGYN motif in several species, enabling in most cases subtle protein variations. Splice donor sites have a highly conserved GT or GC dinucleotide and an extended intronic consensus sequence GTRAGT that reflects the sequence complementarity to the U1 snRNA. Here, we focus on unusual donor sites with the motif GYNGYN .Caenorhabditis elegans and even in Arabidopsis thaliana. Experimental verification of a conserved GTAGTT donor of the STAT3 gene in human and mouse reveals a remarkably similar ratio of alternatively spliced transcripts in both species.While only one GY functions as a splice donor for the majority of these splice sites in human, we provide computational and experimental evidence that 110 (1.3%) allow alternative splicing at both GY donors. The resulting splice forms differ in only three nucleotides, which results mostly in the insertion/deletion of one amino acid. However, we also report the insertion of a stop codon in four cases. Investigating what distinguishes alternatively from not alternatively spliced GYNGYN donors, we found differences in the binding to U1 snRNA, a strong correlation between U1 snRNA binding strength and the preferred donor, over-represented sequence motifs in the adjacent introns, and a higher conservation of the exonic and intronic flanks between human and mouse. Extending our genome-wide analysis to seven other eukaryotic species, we found alternatively spliced GYNGYN donors in all species from mouse to In contrast to alternative splicing in general, GYNGYN donors in addition to NAGNAG acceptors enable subtle protein variations. Given the rather limited number of human genes , alternaThe skipping of an exon is the most frequent alternative splice event, followed by alternative splice donor and acceptor sites . Such spThe recognition of donor and acceptor splice sites is different. While the acceptor AG and its preceding polypyrimidine tract is recognized by the U2AF heterodimer , the donHere we investigate whether alternative splicing at a GT or GC donor dinucleotide 3nt up- or downstream is possible. This type of alternative splicing requires a GYNGYN donor motif (Y stands for C or T) and is of interest because it would result in similar subtle protein changes like at NAGNAG tandem acceptors and thus increase the proteome plasticity. We found expressed sequence tag (EST) and/or mRNA evidence for alternative splicing at 110 human GYNGYN tandem donors and confirm the existence of both splice forms by RT-PCR experiments in seven cases. We report the occurrence of alternative splicing at GYNGYN tandem donors in six other animals and a plant. Analyzing the GYNGYN motifs that do and do not allow alternative splicing, we found significant differences in the stability of the U1 snRNA binding, conserved exonic and intronic flanks between human and mouse, and over-represented sequence motifs in the intronic flanks.Although the great majority of introns begins with a GT dinucleotide, a small fraction of 0.76% begins with GC . To inveBy searching dbEST and the human mRNAs from GenBank, we identified experimental evidence for alternative splicing at 110  tandem donors (in the following we term these tandem donors 'confirmed')  in the vicinity of donor sites lead to a shift in the splice site -26. To cSTAT3 in leucocytes of six individuals and consistently observed both transcripts. This agrees with our in silico finding that tandem donor splicing in general does not depend on specific genotypes and further excludes the possibility that a peculiarity of the spliceosome or its components is the reason for the two splice forms.To further support the EST-derived confirmation of alternative splice events at tandem donor sites, we performed RT-PCR in several human tissues. We selected eight genes with confirmed GYNGYNs having at least three ESTs for e and i transcripts Table . We direThe U1 snRNA determines the donor site by base pairing with the mRNA . To defiNevertheless, there are 17 of the 81 confirmed GTNGTN tandems with more ESTs for the i donor than the e donor. Therefore, we compared the free energy values and found that 15 of these 17 cases (88%) have a lower free energy for the i donor, thus allowing a more stable U1 binding Figure . LikewisSince only a small fraction of all human tandem donors are confirmed, we searched for differences between confirmed and unconfirmed ones. Plotting the free energy values for e and i donors shows that most confirmed GTNGTNs are located at the left part Figure  while unWe assumed that the strength of both donors might be a criterion to distinguish functional from non-functional tandem donors. To test this experimentally, we selected nine unconfirmed GTNGTNs with a low free energy for both donor sites for experimental verification. As for confirmed GTNGTNs, RT-PCR products were directly sequenced and the sequencing traces were inspected for overlapping sequences. For none of the nine candidates, we found evidence for alternative splicing at the tandem donor, suggesting that the majority of unconfirmed GTNGTNs is presumably not alternatively spliced. However, our direct sequencing approach does not exclude that the alternative transcript is expressed at a low frequency.We conclude that: stable U1 binding is necessary but not sufficient for alternative tandem donor splicing; the currently confirmed GYNGYN represent a large fraction of all functional tandem donors; and, in contrast to NAGNAG acceptors , alternaSince the free energy of U1 binding seems not to be the only discriminative criterion, we searched for other differences between confirmed and unconfirmed GTNGTNs. The regulation of alternative splicing often involves auxiliary exonic and intronic splice enhancer and silencer elements  that are bound by trans-acting RNA-binding proteins like serine/arginine rich (SR) proteins and hnRNPs -36. PrevTo find out if specific ISE hexamers are statistically over-represented, we used a resampling strategy. We randomly sampled 10,000 sets, each comprising 81 intron flanks from unconfirmed GTNGTNs. We estimated the P-value as the fraction of random sets with a higher frequency of a given ISE hexamer compared to the observed frequency in confirmed tandem donors. CGGGGT is the only one among the 133ISE motifs that is significantly over-represented in the vicinity of confirmed GTNGTN donors as all 10,000 random sets have a lower frequency . To find out if other sequence motifs are over-represented in the intron flanks of confirmed tandem donors, we repeated this procedure with tetramers. A word length of 4 nucleotides was chosen to account for the rather small data set. We only compared the 119 tetramers that occur at least with the expected frequency in the intron flanks of confirmed GTNGTNs. We found a significant overrepresentation for GGGT and CGGG , while the tetramer GGGG has a corrected P-value slightly above the 0.05 threshold . Since both GGGT and CGGG are substrings of the over-represented ISE CGGGGT, no new sequence motifs were found. The common feature of the over-represented sequence motifs is the G triplet. Interestingly, this motif occurs in 82 of the 133 ISEs  and is aOf the 81 confirmed GTNGTNs, 72 (89%) are located downstream of a coding exon; thus, alternative splicing at these sites results in 3 nucleotide indels into the coding sequence. The effect for the protein depends on the phase of the intron as well as the sequence of the GTNGTN and the upstream/downstream exon. In intron phase 0 (intron location between two codons) the GTN of the i donor is inserted/deleted and codes for a valine. In intron phase 1 and 2 , three different events are possible: indel of a single amino acid; exchange of a dipeptide and a different amino acid; and indel of a stop codon. Of the 72 GTNGTNs, 37 (51%) are located in phase 0, thus a valine indel is the most frequent event at the protein level. Of the 28 (39%) GTNGTNs in phase 1, 18 result in single amino acid events , 8 exchange a dipeptide and an unrelated amino acid and in two cases splicing at the i donor creates a stop codon. The 7 (10%) confirmed GTNGTNs in phase 2 are interesting since they either result in indels of rare amino acids  or insert/delete a stop codon in two cases.FAM65A, NM_024519, exon 21; BRSK2, NM_003957, exon 19; phase 2: ABC1, NM_022070, exon 8; KLHL5, NM_001007075, exon 3). In two cases  the splice form with the premature stop codon is a clear candidate for nonsense-mediated mRNA decay [FAM65A 33 residue deletion at the carboxyl terminus, BRSK2 7 residue deletion).Thus, alternative splicing at four tandem donors has a more drastic impact on the proteins by the indel of a stop codon  and 6 dipeptide exchanges . Compared to the protein events for GTNGTNs, the indel of alanine, glutamine and leucine is only observed for tandems with a GC donor.Next, we compared the frequency of single amino acid events in phase 1 and 2 for confirmed and as a control for unconfirmed GTNGTNs. While only 42%  unconfirmed tandem donors in phase 1 result in a single residue indel, this percentage is significantly higher for confirmed tandems . The small number of phase 2 tandems does not allow a significant result, although the same trend is visible . These findings argue for a preference to insert/delete only single amino acids, presumably because this is a less dramatic event compared to dipeptide exchanges. However, we cannot exclude the possibility that this result is an indirect consequence of a sequence bias of the GTNGTN motif and its context for confirmed tandems that primarily aims at a more stable U1 snRNA binding. For GTNGCN and GCNGTN tandems, only phase 2 donors result in a high percentage of single amino acid indels  while phase 1 events show no bias .Mus musculus), rat (Rattus norvegicus), chicken , zebrafish (Danio rerio), fruitfly (Drosophila melanogaster), and nematode (Caenorhabditis elegans). The percentage of GTNGTN motifs in all donor sites is quite similar in all species and ranges between 2.1% and 3.4%  of the 81 human confirmed GTNGTNs. To assess whether this percentage is high or not, we counted GTNGTN conservation for the 3,909 unconfirmed tandems  and found a very similar percentage of 65.5% . The fraction of tandem donors that have a completely identical GTNGTN pattern in mouse is also equal: 40 of 81 (49.4%) confirmed, 1,939 of 3,909 (49.6%) unconfirmed. Thus, there is no evidence for a general selection pressure to maintain confirmed tandem donors since the divergence of the human-mouse ancestor.STAT3 is conserved in mouse and both e and i transcripts are supported by mouse ESTs. As in humans, we performed RT-PCR in several mouse tissues to further support the EST-derived confirmation of alternative splice events ) of the conserved and confirmed human GTNGTNs is also confirmed in mouse. For example, the GTAGTT donor of intron 21 of s Figure . We founThe intronic flanks of alternative exons are significantly more conserved in mouse compared to the flanks of constitutive exons, a fact which is presumably attributed to the force to maintain regulatory elements . From thSTAT3, thus excluding the possibility of allele-specific instead of alternative splicing [We report the occurrence of alternative splice donor usage for GTNGTN, GTNGCN, and GCNGTN motifs in eight investigated eukaryotic species. Apart from our experimental verification of seven human and one mouse GYNGYN donors, several lines of evidence indicate that the majority of observed events is attributable to real alternative splicing. Firstly, numerous GTNGTNs are confirmed by multiple ESTs/mRNAs and for several of these events both e and i transcripts are deposited in the RefSeq database. Secondly, the existence of orthologous tandem donors that are confirmed in two or more species makes EST artifacts or database errors unlikely. Thirdly, these GTNGTN donors have a higher conservation of the exonic and intronic flanking regions, a situation that is typical for conserved alternative splice events ,46,48,49splicing . FinallyC. elegans, A. thaliana and humans, it is likely that all species that have alternatively spliced genes are able to produce e and i transcripts at certain tandem donor sites. The detection of 44 alternatively spliced tandem donors in A. thaliana is consistent with the finding that alternative splicing in plants is not as rare as thought for a long time [C. elegans [C. elegans is confirmed, which is particularly striking since C. elegans has the highest fraction of HAGHAG acceptors (Table C. elegans has the similar percentage of confirmed tandem donors compared to the other species (Table C. elegans that often lack consensus sequences for the branch point and the polypyrimidine tract [We found that the percentage of donor sites with a GYNGYN motif as well as the percentage of tandem donors that are confirmed is very similar between the eight investigated species (tolerating some variation probably due to differences in the number of ESTs and mRNAs). Given the large evolutionary distance between ong time ,51. Prev elegans . To comp elegans ,14 to thrs Table . This rane tract .Although only a fraction of the tandem donors is confirmed, we found features that distinguish confirmed from unconfirmed ones. Since the non-annotated donor of unconfirmed tandems does not allow a sufficiently stable binding to the U1 snRNA, the other donor is used exclusively in the splice process. For confirmed tandem donors, both sites allow a stable binding to U1 snRNA. However, in most of the confirmed cases one donor has a better strength and this results in its preferred usage as measured by the EST ratio between both transcripts. The second discriminative feature is the overabundance of G triplets in the intronic flanks of confirmed GTNGTNs, especially for introns shorter than 200 nt. This triplet is the core of many known ISE motifs ,40 and wFGFR1 gene [Most confirmed GYNGYNs have a low free energy of U1 snRNA binding to both the e and i donor, suggesting that the U1 snRNA can stably bind to both sites. However, there are a few exceptions where one donor is much stronger than the other one in a confirmed tandem Figure . The mecFR1 gene . MoreoveFR1 gene ,55. FurtFR1 gene ,57. We bFAM3B (NM_206964), the verified SNP rs417708 results in two alleles, GTGGTA and GCGGTA. For the C allele, the free energy of the i and e donor is -3.4 and -6.2 kcal/mol, respectively, while this value is more balanced for the T allele . This agrees well with the prediction of the splice site analysis server [PEG3 gene . Both homozygous G genotypes and heterozygous genotypes with a maternally inactivated T allele will result in an exclusive splicing at the i donor.Previously, we found that the impact of SNPs in NAGNAG acceptors on alternative splicing can be accurately predicted . Therefos server ,59. ThusBRUNOL4 (NM_020180), for which we found 14 e-E, 3 i-E and 6 e-I transcripts in dbEST that result in protein forms with a GPA, AA, or GP peptide, respectively.Higher eukaryotes typically express multiple transcripts and proteins from a single gene. A prominent mechanism is alternative splicing as about 74% of the human multi-exon genes express more than one splice variant . While mSTAT3 GTNGTN donor is strikingly equal in human and mouse. In light of the discussion about functional versus non-functional alternative splicing [ALDH18A1 results in an isoform that is insensitive to ornithine inhibition [Despite many GYNGYN donors, we found only a minority that allows alternative splicing. Nevertheless, among the human confirmed and evolutionary conserved tandem donors we found a significant fraction to be confirmed in other species. Moreover, the splicing pattern of the splicing ,62, thissplicing ,64. Intehibition . Furtherhibition -18. Thushibition .M. musculus, rn3 (June 2003) for R. norvegicus, galGal2 (February 2004) for G. gallus, danRer2 (June 2004) for D. rerio, dm2 (April 2004) for D. melanogaster, and ce2 (March 2004) for C. elegans. For A. thaliana, we used the genome assembly  to build a list of donor sites according to the CDS feature annotations in GenBank format and screened this list for donor sites with a GYNGYN sequence pattern.We downloaded from the UCSC Genome Browser  the HumaFor checking if a GYNGYN donor is confirmed (at least one EST/mRNA for the e as well as i transcript), we compiled two search strings: 30 nucleotides from the upstream exon and 30 nucleotides from the downstream exon for the i transcript; and 30 nucleotides from the upstream exon, the GYN of the i donor and 30 nucleotides from the downstream exon for the e transcript. Then, we used BLAST against all ESTs and mRNAs for the respective species, allowing at most one mismatch or one gap but demanding exact identity for the region 27-33 for i-transcripts and 27-36 for e-transcripts. The ESTs were downloaded from the UCSC Genome Browser . mRNAs were downloaded from GenBank at the same date as the ESTs. For acceptor sites with a NAGNAG pattern, we repeated the analysis using analogous procedures and the same data described above.Human-mouse genomic alignments (hg17-mm6) were downloaded from the UCSC Genome Browser . We used the genomic position of human and mouse donor sites to select the respective alignment chain. From the alignments, we determined whether a human GTNGTN donor is conserved  or completely identical. For the per-position identity computation, we considered the alignment part up to 100 positions upstream and downstream. For each position, we counted how often there is identity between human and mouse (Nid), and how often there is a mismatch or gap (Nmm). The per-position identity value is Nid/(Nid+Nmm). Alignment positions with an 'N' aligned to a nucleotide were ignored.To find tandem donors that are orthologous and confirmed in human and mouse, we used BLAST with the human-confirmed search strings against the search strings of the mouse-confirmed GTNGTNs. Furthermore, we used BLAST with the human-confirmed search strings against the mouse ESTs and mRNAs. Using the UCSC and Ensembl genome browser, we manually checked each hit with an E-value of less than 1e-3 for being alternatively spliced in both species and for having a true orthologous relationship.We extracted a 9 nucleotide genomic context (3 nucleotides upstream to 3 nucleotides downstream of the GYN) for the e and i donor of confirmed and unconfirmed GYNGYNs. The free energy and number of base pairs with the U1 snRNA were computed according to  with theWe extracted the genomic sequence 100 nucleotides upstream (exonic) and 100 nucleotides downstream (intronic) of GTNGTN donor motifs. To identify over-represented ISE hexamer motifs, we used a resampling procedure to estimate the P-value for a higher frequency in the intronic flanks of confirmed GTNGTNs. To this end we randomly sampled 10,000 sets of 81 intronic flanks of unconfirmed GTNGTNs and computed the frequency for each of the 133 ISE motifs in the 10,000 random sets. The P-value for one ISE is the fraction of random sets with a higher frequency compared to the observed frequency for confirmed GTNGTNs. To correct for multiple testing, each P-value is multiplied by 133. For the general search for over-represented motifs, we decided to use tetramers (word length 4 nt) instead of hexamers since the dataset of the confirmed tandems is rather small. Since we were searching for over-represented motifs in the intronic flanks of confirmed GTNGTNs, we expected that such motifs occur at least with the expected frequency under a null model and with a significant higher frequency compared to the flanks of unconfirmed GTNGTNs. There are 97 overlapping tetramers in a 100 nucleotide sequence, thus we analyzed a total of 81 × 97 = 7,857 tetramer occurrences. For complete random sequences, each tetramer should occur 7857/256 = 30.7 times. Since intron sequences are not random, we found a total of 119 tetramers that occur 30 times or more in the flanks of confirmed GTNGTNs. For these 119 tetramers, we repeated the procedure described above but multiplied the P-value by 119.TOM1 ; STAT3 ; ANAPC4 ; RBM10 ; ANGPT1 ; SEMA5B ; CXorf44 ; and LTBP1 . In addition, Stat3 of M. musculus was analyzed in the respective Clontech mouse tissue panel by amplifying and sequencing the homologous region with the oligonucleotides GCCATCCTAAGCACAAAGCC and GGCTCAGCACCTTCACCGTT. All sequence traces have been deposited in the NCBI Trace Archive . To estimate the relative amounts of e and i transcripts of human and mouse STAT3, we cloned the respective amplicons into pCR2.1-TOPO  and propagated the clones in Escherichia coli TOP10 cells. Plasmids were isolated from several isolated clones and their inserts were sequenced using universal M13 primers.Eight genes with multiple EST evidence for alternative splicing at a tandem donor were analyzed by RT-PCR in different tissues by using cDNA from multiple tissue cDNA panels  as PCR templates. Primers were designed for the exons flanking the tandem donor with distances to these donors that allow reliable amplification and sequencing. PCR was performed in a total volume of 25 μl using ReadyToGo PCR beads  with 5 pmoles of each primer and 1 μl of cDNA. Cycling conditions were 94°C for 30 s followed by 35 cycles with 94°C for 20 s, 57°C for 30 s and 72°C for 30 s, followed by a final extension at 72°C for 10 minutes. Amplified fragments were precipitated with ethanol and ammonium acetate, washed with ethanol and sequenced using DyeTerminator chemistry  and the respective PCR primers on a 3730 xl DNA Analyzer (Applied Biosystems). Genes and their primer sequences were: A2ML1 ; TMEM63C ; RNPC3 ; GOLGA3 ; ART5 ; KIAA1853 ; K5B ; SAMD4B ; and LOC221711 .The same strategy was applied to a set of genes with unconfirmed tandem splice donors. Genes and their primers were: To facilitate further experimental and computational studies of tandem splicesites, we recently developed a database, TassDB , which pThe following additional data are available with the online version of this paper. Additional data file Data for all human confirmed GYNGYN splice donor sites identified in this studyClick here for fileData on the strength of e and i human GYNGYN donorsClick here for fileConfirmed GYNGYN donors for seven speciesClick here for fileSelected sequence traces that exemplify the experimental verification of GYNGYN donorsClick here for fileControl experiments for detecting minor splice forms by direct sequencing of RT-PCR productsClick here for file


Previously, a total of five breast cancer subtypes have been identified based on variation in gene expression patterns. These expression profiles were also shown to be associated with different prognostic value. In this study tumour samples from 27 breast cancer patients, previously subtyped by expression analysis using DNA microarrays, and four controls from normal breast tissue were included. A new MetriGenix 4D™ array proposed for diagnostic use was evaluated.We applied MetriGenix custom 4D™ arrays for the detection of previously defined molecular subtypes of breast cancer. MetriGenix 4D™ arrays have special features including probe immobilization in microchannels with chemiluminescence detection that enable shorter hybridization time.The MetriGenix 4D™ array platform was evaluated with respect to both the accuracy in classifying the samples as well as the performance of the system itself. In a cross validation analysis using "Nearest Shrunken Centroid classifier" and the PAM software, 77% of the samples were classified correctly according to earlier classification results.The system shows potential for fast screening; however, improvements are needed. An array platform  was introduced in which molecular interactions occur within three-dimensional volumes of ordered microchannels rather than at a two-dimensional surface . The micfied RNA ,3. The pMolecular signatures based on gene expression patterns of breast carcinomas, specifying different subtypes of tumours, have been identified by the use of DNA microarrays -7. SpeciGene expression patterns from 269 genes selected to optimally define the different subtypes of breast cancer were analyzed in breast tumours and normal tissue from 31 individuals . The aim of this pilot study was to investigate whether this type of arrays would have the potential as a diagnostic tool for molecular classification in a clinical setting.Here we show that the MetriGenix 4D™ arrays with a limited number of carefully selected probes, perform similarly to other microarray platforms, although technical difficulties were experienced with the prototype system.In total, 27 breast tumour samples and 4 normal tissue samples were analyzed in this study. Among the tumour samples, 20 biopsy tissues from early breast carcinomas were included of which ten have been previously sub-classified as luminal subtypes, the other half as non-luminal subtypes using traditional two-dimensional microarray platforms such as Stanford cDNA arrays, Agilent Human Whole Genome Arrays and Applied Biosystems Human Genome Survey Microarrays . Further®  as described by the manufacturer. The RNA quality was evaluated by microcapillary electrophoresis using an Agilent 2100 Bioanalyzer  and concentration measured by using NanoDrop .Total RNA was extracted from fresh frozen tissue samples by using TRIzolWe selected 269 genes that best represented the classification scheme in breast cancer to be synthesized and immobilized on the MetriGenix-Chip . Double-stranded cDNA was purified in Phase-Lock Gel Tubes  and in vitro transcribed by Ambion's MEGAscript ™ T7 High Yield Transcription kit  followed by cleanup with RNeasy® RNA isolation kit columns (Qiagen). Amplified cRNA was evaluated on the Agilent 2100 Bioanalyzer (Agilent Technologies). The cRNA was biotin-labelled using MetriGenix Bio ULS  (0.5 Units/μl) in a one-step chemical coupling reaction at 85°C for 30 min .Total RNA was amplified using a two-step cRNA synthesis scheme typical for microarray experiments. First strand cDNA was synthesized by annealing T7-(T) 24- primer (100 pmol/μl) with 5 μg total RNA in a final volume of 12 μl at 70°C for 10 min, followed by addition of first-strand master mix  to a final volume of 20 μl. The reaction was incubated at 42°C for one hour. Second strand synthesis followed immediately by adding 5× Second Strand Buffer, 10 mM/each dNTPs mix, 10 U/μl Prior to hybridization the biotin-labelled cRNA were mixed with spike-in controls , Sample Dilution Buffer 2 (MetriGenix) and herring sperm DNA (Invitrogen) and denatured for 5 min at 90°C. The sample was then injected into the sample compartment of the 4D array, along with blocking and staining reagents into their respective compartments.Custom 4D arrays to monitor the genes of interest were supplied by MetriGenix . For each gene, a 50- to 60-mer probe was designed based on publicly available sequences and to have GC content in the range of 45 to 55 percent and a melting temperature between 64 and 68°C. For product quality control (QC) the following steps were performed: First, hybridization was performed with just the complements to the control probes to confirm that there was no cross-contamination of probes on the chip. Second, a test cRNA was hybridized to the chip in the absence of the control targets; since the controls were bacterial and the test cRNA mammalian, no hybridization was observed in the control probes (otherwise the chips failed QC). Third, control targets were added to every cRNA that was hybridized to the chip and the intensity of the spots was used qualitatively to confirm the hybridization results. Probes were synthesized with a 5' amino modification and printed on MetriGenix arrays using a Gene Machine Omnigrid arrayer. The arrays are housed in a 4D cartridge that includes reagent reservoirs and interfaces with the MGX2000 and MGX1200CL array processing stations.4D array hybridizations were performed on the MGX™ 2000 hybridization station, which controlled all subsequent steps . After four hours of hybridization , the chip was placed in the MGX 1200 CL detection unit for chemiluminescence (CL) detection with exposure times usually ranging from 2 to 5 s. Subsequent image analysis was performed with the MetriSoft software (MetriGenix) that generated an excel file containing the experiment data for subsequent analysis.The Metrisoft software operated on two different concepts, the noise floor and a stringent threshold value, to filter spots in the individual chip analysis. The noise floor was a value calculated by the software in each individual chip analysis that related to the amount of noise in the chip and which was subsequently used to determine the threshold value. The stringent threshold value was calculated as 3 times the noise floor, an empirical estimate of an 'absent' spot based on the image noise. Any signal below this value was not considered significant and assigned to the threshold value. For intra-chip normalization, the signal intensity of each individual spot was divided by the threshold to produce the normalized values within each chip. Data from 3 successful hybridized controls  were averaged for each gene to obtain a mean expression value. Next, to create log-transformed (base 2) pseudo ratios the value of each sample was divided by the mean of the three controls for every gene.Principal Component Analysis (PCA), hierarchical clustering and ANOVA were performed by using Avadis Prophetic software . Data were mean-centered, clustered using Euclidean distance measures and visualized using a heat map in which numeric values are represented in colour intensities . For ANOVA, samples were assigned class designations and gene expression data were analyzed assuming equal variance. Data were ranked based on p-values and F-statistics. In addition, a set of genes that best discriminated the two identified main subtypes of breast cancer were determined using "Nearest Shrunken Centroid classifier" and the PAM software . For thiThe aim of this study was to demonstrate whether these novel arrays would have the potential to be used in molecular classification in a diagnostic setting in a future implementation. The limited number of MetriGenix chips available in this study allowed us to successfully hybridize and analyze altogether 25 of 31 samples . Therefore, in the further evaluation with respect to the accuracy in classifying the samples we concentrated on the luminal  vs. non-luminal  groups instead of all five subtypes.ESR1, GATA3, XBP1, MUC1 while the basal-like tumours showed no expression of these genes. Further, the luminal cluster was divided into four sub-groups corresponding to the luminal A (dark blue), luminal B (light blue), the latter with less expression of luminal epithelial specific genes expressed, the previously identified ERBB2+ - (purple) and the normal-like group (green). Although there were few samples analyzed in this study, those representing each subtype grouped together.To evaluate the data generated by the MetriGenix analyses, an ANOVA analysis was performed excluding the control samples to identify the genes that best separated the samples into two groups; luminal and non-luminal see . Genes wTo test whether the clustering pattern could be visualized by using data from all the 226 "intrinsic" only genes printed on the 4D array, a hierarchical clustering was performed see . As prevTo further evaluate the dataset, prediction of tumour subtypes was performed using the PAM software and the results are shown in figure Principal component analysis (PCA), based on all genes on the 4D array, confirmed the groupings of samples seen in the hierarchical clustering analysis figure . Again, The sample preparation procedure including two-step cRNA synthesis and hybridization/detection was accomplished in three days. An advantage of the system was the short hybridization time of four hours; however, the multiple-day sample preparation protocol makes the overall analysis not much shorter than other microarray platforms. The system throughput was limited to a single chip per hybridization unit and the handling and purification was difficult due to small exterior buffer reservoirs and small buffer tubes, leading to contamination concerns. Moreover, it was difficult to trace errors, both on the hardware system and the Metrisoft software, as the system lacked an error message function. For more extensive applications, the image analysis software will require significant refinement. For example, artefacts such as pinspots and high background on the sample images occurred that led to the rejection of several arrays from further analysis. In addition, when analysing the images in the Metrisoft software, we consistently observed that the spot-finder did not find all of the spots on the image and manually re-reading of the image was necessary.We conclude that by selecting only the data from the well performed arrays (25/31) and by using key signature genes for breast-cancer subtypes immobilized on the MetriGenix 4D™ array, we were able to classify samples into the same subtypes as they were previously classified by using other microarray techniques, with a relatively high probability (23% misclassification). With higher throughput and improved performance this microarray platform has the potential to be a valuable tool for rapid routine gene expression profiling.DB and AS are previous employees of MetriGenix. MetriGenix is a private company that manufactured the arrays employed in this study, which was performed on a collaborative basis. All other authors declare that they have no competing interests.AM was involved in laboratory experiments, analyzing the data and drafted the manuscript. HJ was involved in laboratory experiments. DB carried out the statistical computation of the data. AS were involved in data analysis. PEL and BN were involved in providing breast cancer samples. TS and ALBD initiated and designed the study and were involved in writing the manuscript.The pre-publication history for this paper can be accessed here:Genes-on-chip. A listing of the genes immobilized on the 4D array along with sequence information of each probe.Click here for fileANOVA-analysis output. ANOVA analysis of all the genes immobilized on the MetriGenix breast cancer chip.Click here for fileHierarchical clustering of "intrinsic" only genes. Hierarchical clustering diagram of the 226 "intrinsic" only gene subset immobilized on the MetriGenix chip. Coloured branches represent the different subtypes as previously determined using a different DNA microarray platform [platform ; Dark blClick here for filePAM-analysis output. PAM output of the most differentially expressed genes between the two groups: luminal and non-luminal.Click here for file


The primary efficacy variable was the average FEV1 from the first intake of study medication to the measurement at 90 minutes. Secondary endpoints included changes in FEV1 at other timepoints and change in respiratory rate at 180 minutes. Treatment success, treatment failure and patient assessment of the effectiveness of the study medication were also measured.In a 3 hour, randomized, double-blind study, a total of 115 patients with acute asthma (mean FEV1 increased after administration of the study medication in both treatment groups. No statistically significant difference between the treatment groups was apparent for the primary outcome variable, or for any of the other efficacy endpoints. There were no statistically significant between-group differences for treatment success, treatment failure or patient assessment of medication effectiveness. Both treatments were well tolerated.FEV2-agonist.Budesonide/formoterol and formoterol provided similarly rapid relief of acute bronchoconstriction in patients with asthma who showed evidence of refractoriness to a short-acting β Oral prednisolone  was administered to all patients 90 minutes after they received the second dose of study medication.The study design is shown in Figure 1 from the first intake of study drug to the measurement at 90 minutes. The following were assessed as secondary efficacy variables: the change in FEV1 at t = 3, 15, 60, 90 and 180 minutes after the last intake of study medication; the change in respiratory rate at 180 minutes; the proportion of patients with treatment success at 90 and 180 minutes; the proportion of patients with treatment failure at 180 minutes; and the effectiveness of the study medication at 3 and 15 minutes.The primary efficacy variable was the average FEV1 was measured by spirometry according to European Respiratory Society (ERS) recommendations [FEVndations . RespiraThe incidence, severity and type of adverse events occurring during the study were recorded. Deterioration of asthma was not recorded as an adverse event unless it caused the patient to withdraw from the study or was classed as serious by the investigator. A serious adverse event was defined as an event that: caused death or was immediately life-threatening; required inpatient care or prolongation of hospitalization; resulted in persistent or significant disability or incapacity; or required medical intervention to prevent one of these outcomes.Measurements of serum potassium, pulse rate, blood pressure, electrocardiogram (ECG) variables, and oxygen saturation were also used to assess safety. The outcome variables for serum potassium were the minimum and average values after administration. Blood samples for the determination of this variable were obtained at baseline (t = -15 minutes) and 30, 90 and 180 minutes after administration of the last dose of study medication.1 from the first intake of study drug to the 90 minute measurement .A sample size of 50 patients per group was required to have an 80% chance of detecting a difference of 12% between treatments in average FEV1 were analysed using multiplicative analysis of variance (ANOVA), with treatment and country as fixed factors and the pre-administration value (t = -5 minutes) as a covariate. The change in respiratory rate was analysed using additive ANOVA. The proportions of patients with treatment success, treatment failure and reports of effective medication were analysed using logistic regression models with treatment and country as factors. Odds ratios were estimated and described with 95% confidence limits. Clinical laboratory data, vital signs and physical measurements relating to safety were compared between treatment groups with an additive ANOVA model. Adverse-event data were evaluated using primarily descriptive statistics.Efficacy analyses were performed on the full analysis set; all patients who received at least one dose of study medication were included in the safety analysis. Changes in FEVPatients were enrolled and treated between April 2002 and August 2003. A total of 277 patients were recruited into the study, 115 of whom fulfilled the inclusion criteria and were randomized to receive treatment with either budesonide/formoterol (n = 58) or formoterol (n = 57). One patient, who was randomized into the formoterol group, discontinued the study as a result of an adverse event. The full analysis set comprised all randomized patients.1 was 1.13 L . After treatment with salbutamol 400 μg, the mean reversibility in FEV1 was 5.6% of the baseline value .The two treatment groups were comparable at baseline, in terms of both demographics and other clinical characteristics Table . Patient1 increased after study drug administration in both treatment groups  from the first intake of study drug to the 90-minute measurement was 17.4% in patients receiving budesonide/formoterol and 16.5% in formoterol-treated patients. There was no significant difference between the groups for the primary outcome variable.FEVs Figure . The ave1 at any of the timepoints analysed over the full 180-minute assessment period, or for the maximal FEV1 value achieved  compared with those being treated with formoterol . Despite the initially poor response to salbutamol in all patients, only a small minority of treatment failures were seen in both groups . The majority of patients described their medication as effective. After 3 minutes, 97% of patients receiving budesonide/formoterol considered their medication to be effective; this figure remained at 97% at 15 minutes. The corresponding values for the formoterol treatment group were 93% and 96%, respectively.There were no statistically significant between-group differences for any of these variables.Nine patients in each treatment group (16%) reported a total of 24 adverse events. There were 13 adverse events in patients receiving budesonide/formoterol and 11 in the formoterol treatment group. These events were mostly mild or moderate in intensity. Tremor and headache were the most frequently reported adverse events and there were no differences between the treatment groups in the incidence of patients reporting these symptoms (Table One patient in the formoterol group discontinued the study after 2 hours because of worsening asthma. A patient in the budesonide/formoterol group suffered a serious deterioration in asthma symptoms but managed to complete the study. These two episodes were reported as serious adverse events (asthma aggravated). Neither was considered to be causally related to the study drug. There were no deaths during the study.Treatment with both budesonide/formoterol and formoterol was well tolerated and systemic effects were similar for both treatments. The majority of patients in both treatment groups had normal serum potassium before treatment and at the end of the study period. The mean values for the average serum potassium during the 180 minutes after study drug administration were 3.82 and 3.88 mmol/L for the budesonide/formoterol and formoterol groups, respectively. The corresponding mean pre-dose values were 3.83 and 4.01 mmol/L, respectively. Abnormally low values (<3.0 mmol/L) were reported for two patients 180 minutes after the last intake of study medication; both of these patients were in the formoterol group. The lowest treatment value recorded for a patient in the budesonide/formoterol group was a transient decrease to 2.7 mmol/L after 90 minutes; the lowest value for formoterol-treated patients was 2.6 mmol/L, which occurred in a patient with a pre-dose value of 2.4 mmol/L. In the formoterol group, one case of hypokalaemia was reported as an adverse event. Overall, the effects of budesonide/formoterol and formoterol on serum potassium were small and were not considered to be clinically important; there were no statistically significant differences between the treatment groups with regard to this parameter.Vital signs, ECG parameters and oxygen saturation also showed only small changes over time during the course of the study. There were no statistically significant differences between the treatments for the average or maximum/minimum values for any of these variables, with the exception of heart rate, for which budesonide/formoterol had a lower maximum value (mean of 91.6 beats per minute) than formoterol .2-agonist, salbutamol. There was no difference between the two treatments with regard to their efficacy and tolerability over the 180-minute assessment period. At the end of the study, a high proportion of patients in both groups had been successfully treated.Patients with acute asthma require efficacious medication to reduce bronchoconstriction. In this study, budesonide/formoterol and formoterol proved similarly safe and effective for the treatment of patients with acute asthma who had not initially responded adequately to the short-acting β2-receptor response in patients receiving therapy with ICS, as has been described previously [1 of only 5.6% from baseline), they may not have been sufficiently refractory to demonstrate the potential benefit provided by ICS. This is suggested by the rather good initial responses  in the patients who received formoterol alone.Previous studies have demonstrated that formoterol is at least as effective as salbutamol for the treatment of acute exacerbations ,6 and iteviously ,11,17. A2-agonists in acute asthma is complicated by both conceptual and methodological problems. Among the former are the difficulty in distinguishing between β2-agonist refractoriness caused by downregulation – a feature that has been confirmed in vitro [in vivo – and a lack of clinical response caused by pathological changes in asthmatic airways  and chronic structural changes (such as airway remodelling) [2-agonist receptor density on circulating monocytes, together with other in vitro tests, may have been useful, but the results of such testing would not have been available in time to assist with patient selection for this acute study. A minority of patients were being treated with ICS at the time of their exacerbation, but most had a history of previous exacerbations, suggesting that many patients' asthma had been chronically undertreated. In addition, for 78% of the patients the acute symptoms had lasted for >24 h before study entry and so it is possible that, in many patients, airway pathology may have been well established. Consequently, airway pathology, rather than downregulation of β2-agonist receptors, may have accounted for β2-agonist refractoriness.The identification of patients with refractoriness to βin vitro , but notdelling) ,19. Assa2-agonists. Patient recall of β2-agonist use is often poor. Moreover, we waited only 15 minutes after administering salbutamol 400 μg before patients were treated with the study drug. This was necessary because patients were distressed and it was considered neither safe nor compassionate to await the peak effect of the salbutamol (between 15 and 30 minutes after administration) to exclude patients who responded more slowly. In contrast, patients were followed for longer periods after receiving the study drugs. There was, however, some evidence that the initial response to salbutamol was already at a plateau or in decline when the study drugs were administered, although there was no evidence of a loss of effect with either of the study drugs during the 3-hour assessment  versus placebo. While an improvement in lung function was evident 1 hour after drug administration, the effects increased over time to become statistically significantly different compared with placebo at 3–4 hours [Pansegrouw  is not a–4 hours . In the 2-agonist combination (at the dose administered in the present study) is effective in preventing severe exacerbations if given very early, when the symptoms of loss of asthma control first become apparent, but may be less beneficial for patients in whom an exacerbation has become established. In our study investigating symptom relief in patients with acute asthma, the long-acting β2-agonist alone was as effective as the combination therapy – even in patients presenting for emergency treatment who had used their short-acting β2-agonist liberally and appeared to be refractory to β2-agonist therapy, as judged by poor bronchodilatory response to short-acting β2-agonists.It is also possible that the dose of budesonide used in this study was too low. In patients who deteriorate whilst on regular doses of ICS, doubling the maintenance dose when symptoms of an exacerbation become apparent has not been shown to be effective in preventing the exacerbation ,22, but In addition to being effective, both budesonide/formoterol and formoterol were safe and well tolerated in the present study. The patterns of adverse events raised no safety concerns and, overall, the changes over time in systemic effects were very minor. These results are in line with those from earlier studies. Ankerst and colleagues  have repIn conclusion, budesonide/formoterol and formoterol provide rapid relief of acute bronchoconstriction in patients with asthma, producing effects of a similar magnitude. Both preparations are well tolerated.


Twelve promoters of genes differentially expressed between humans and chimpanzees were tested for expression activity in culture cells. Seven promoters showed a significant difference in expression level between the human and chimpanzee promoter, but only three were in the same direction as the tissues, indicating that relevant expression differences between humans and chimpanzees will be difficult to predict from cell culture experiments or DNA sequences It has long been argued that changes in gene expression may provide an additional and crucial perspective on the evolutionary differences between humans and chimpanzees. To investigate how often expression differences seen in tissues are caused by sequence differences in the proximal promoters, we tested the expression activity in cultured cells of human and chimpanzee promoters from genes that differ in mRNA expression between human and chimpanzee tissues.Twelve promoters for which the corresponding gene had been shown to be differentially expressed between humans and chimpanzees in liver or brain were tested. Seven showed a significant difference in activity between the human promoter and the orthologous chimpanzee promoter in at least one of the two cell lines used. However, only three of them showed a difference in the same direction as in the tissues.Differences in proximal promoter activity are likely to be common between humans and chimpanzees, but are not linked in a simple fashion to gene-expression levels in tissues. This suggests that several genetic differences between humans and chimpanzees might be responsible for a single expression difference and thus that relevant expression differences between humans and chimpanzees will be difficult to predict from cell culture experiments or DNA sequences. Thirty years ago, King and Wilson  proposedcis- and trans-acting factors are involved in transcriptional regulation [trans-acting factors are clearly important, allelic DNA sequence variation in several human promoters has been shown to profoundly influence transcriptional activity [The regulation of gene expression is a complex process involving chromatin structure, DNA methylation, transcription initiation, alternative splicing, RNA degradation, translational control, and posttranslational modifications ,11. Howegulation -21. Althactivity -26. Furtactivity .in vitro promoter activity and mRNA levels in tissues of the organisms.To estimate what fraction of mRNAs differently expressed between human and chimpanzee tissues may be caused by DNA sequence differences in core promoters, we analyzed the activity of human and chimpanzee promoters from 12 genes that differ in their mRNA expression between the two species in brain and liver as measured by microarrays . In eachGene-expression data measured with Affymetrix U95A arrays from livers and the prefrontal cortex of the brains of three humans and three chimpanzees  were useThis left us with human and chimpanzee promoters from 12 genes. From each promoter, a fragment covering approximately 1,500 base pairs (bp) upstream and ≤ 500 bp downstream of the transcription start site without including the start codon was cloned in a plasmid in front of a firefly luciferase reporter gene. For each species, three independent clones were isolated and the insert of each clone was sequenced in its entirety. Each plasmid was mixed with a plasmid containing a sea-pansy luciferase reporter gene under the control of a constitutive promoter and transfected into a human neuroblastoma cell line and a human cervical carcinoma cell line, respectively. Experiments were performed in triplicate and the activities of the two luciferases were measured. All constructs showed an activity at least ninefold higher than the promoter-less control vector in at least one of the two cell lines. To control for transfection efficiency, the measurement of the firefly luciferase was normalized to the measurement of the sea-pansy luciferase and the difference in activity between the three human clones and the three chimpanzee clones analyzed.p-value < 0.05) in both cell lines whereas five  show a significant difference in only one of the two cell lines. Five promoters show no significant difference in either cell line. The average sequence divergence  nor for 220 bp around the transcription start site (p = 0.43) in which most of the conserved regulatory motifs are found [Alu insertion, but does not show a significant difference in its activity in either of the two cell lines.The results are summarized in Figure re found . One proThree promoters  show activity differences in the promoter assays that go in the same direction as the expression differences of the corresponding genes in the tissues. Interestingly, the two promoters  that show qualitatively similar differences in the two cell lines are both in concordance with the tissue expression differences. For four promoters  that show differences in only one of the cell lines, the difference goes in the opposite direction to the expression differences in the tissues.cis-acting variation in mice [in vivo is needed.We have compared the transcriptional activity of human and chimpanzee promoters from 12 genes that differ in their expression levels between humans and chimpanzees in tissues. We find that seven of the 12 promoter pairs differ significantly in their transcriptional activity in at least one of the two cell lines used  go in the same direction in at least one of the cell lines as they do in the tissues, whereas they go in the opposite direction for four genes  in one of the two cell lines. One possible explanation is that the expression differences observed in tissues are due to differences in environmental factors between the species. This, however, seems unlikely, given the high reproducibility of expression differences between studies  that usein vitro. This agrees with the observation that many gene-expression differences are inherited as quantitative traits, that is, several genetic loci are responsible for allelic gene expression differences observed among humans and among mice [even-skipped stripe 2 enhancer in two Drosophila species [A remaining possibility is that additional genetic differences between humans and chimpanzees outside the proximal promoters are numerous and/or strong enough to lead to gene-expression levels in tissues that are often qualitatively opposite to what would be inferred from the activity of the proximal promoter ong mice ,21,35. Iong mice . A probaong mice . When th species .in vitro and the corresponding tissue mRNA levels also holds true for genes that do not show an expression difference between human and chimpanzee tissues. If many genetic differences do indeed influence the expression of a single gene, the proximal promoters of these non-differentially expressed genes would be expected to differ in their activity almost as frequently as the promoters of differentially expressed genes.It is worth noting that the genes studied here are all differentially expressed in human and chimpanzee tissues. It is still unclear to what extent the discordance between behavior of promoters Our results imply that although many promoters may differ in activity between humans and chimpanzees, it will be difficult to predict physiologically relevant gene-expression differences from promoter activities observed in cell lines, even between two closely related species such as humans and chimpanzees. Further work is necessary to elucidate to what extent this applies also to allelic DNA sequence differences in promoters observed within a species. Further work is also needed to elucidate whether a general paradigm for how genome structure translates to gene expression activity can be derived.p-value < 0.05 or > 0.95 (two-sided test) and with a fold-change magnitude of at least 1.4-fold. The false-discovery rate was determined by applying these selection criteria to 10,000 permutations of the original dataset with randomly assigned sample labels. Expression differences were confirmed by masking all probes showing inconsistent hybridization patterns in the two species using a custom mask file as described elsewhere [Genes for promoter analysis were selected on the basis of a large-scale transcriptome comparison between three humans and three chimpanzees in brain and liver using Affymetrix HG U95A and HG U95Av2 arrays . All miclsewhere .BglII or XhoI (depending on the presence of restriction sites in the promoter sequence) were added to the 5'-end of the primer for cloning. Using these primers , we were able to amplify and isolate three independent clones from both species for 12 genes.We used the DBTSS databases of transcriptional start sites  (August Pfu DNA polymerase (Stratagene), using the following conditions: 1 min at 96°C, 45 sec at 96°C, 45 sec at 61°C, 5 min at 72°C for 38 cycles, followed by a final extension at 72°C for 10 min, on Tetracyclers (MJ Research).DNA from one human and one chimpanzee was amplified using Expand 20 kb Plus PCR system (Roche) according to the manufacturer's protocols with an extension time of 3 min, or BglII (NEB) or XhoI (NEB), purified on 1% low-melting agarose gels (Promega) and isolated using QIAquick gel purification kit (Qiagen). These fragments were cloned upstream of the firefly luciferase gene into the BglII or XhoI site of the pGl3 vector (Promega), using T4 Quick Ligase (NEB) and One Shot Top 10 F cells (Invitrogen). Colonies were picked and heated in 10 μl water for 5 min at 96°C, and 2 μl was used as template in a 25 μl PCR reaction, using one primer in the vector (GL2) and one primer in the promoter.PCR products were purified up by QIAquick eight-well cleanup kit (Qiagen), digested with either Positive clones were grown in 7 ml LB medium (Invitrogen) containing 100 μg/ml ampicillin (Sigma) at 37°C overnight. Vector DNA was isolated using a Miniprep kit (Qiagen), and DNA concentration was measured on a Nanodrop UV spectrophotometer (NanoDrop Technologies). All inserts were sequenced  using Big Dye Terminator chemistry (Applied Biosystems).2 and 100% humidity, grown for 20 h and then lysed in 100 μl lysis buffer (Promega).The human neuroblastoma cell line (SHEP ) was obtA 5 μl sample of lysate was used in a Dual-Luciferaser Reporter Assay System (Promega) in a Wallac Victor 2 Luminometer (PerkinElmer). Promoter activity was measured by normalizing the luciferase activity of the promoter constructs to the sea-pansy luciferase activity of the control plasmid from the same well . We assessed the significance (p < 0.05) of different activity in human and chimpanzee promoters using a multi-way ANOVA including species, clones, and replicates.p-values (MAS 5.0) averaged over the 36 pairwise comparisons between human and chimpanzee samples [2 ratio  for the probe sets are given. p-values close to 1 suggest a higher expression in chimpanzees, as does a negative slr. Additional data file Additional data is available with the online version of this paper. Additional data file p-values (MAS 5.0) averaged over the 36 pairwise comparisons between human and chimpanzee samples [2 ratio  for the probe sets are given. p-values close to 1 suggest a higher expression in chimpanzees, as does a negative slr.Supplementary Table Click here for fileSupplementary Table 2: The primer sequences used to amplify the promoter fragments are given here. Primer sequences are written 5' to 3' and lower-case letters indicate added restriction sites.Click here for fileSupplementary Table 3: The normalized promoter activities are listed; that is, the measured luciferase activity of the promoter constructs divided by the sea-pansy luciferase activity, for each of the three replicates that were done for each of the three clones for humans and chimpanzees in the neuroblastoma cell line and the cervix carcinoma cell line, respectively, for the 12 genes analyzed.Click here for fileThe nucleotide sequences of the cloned promoters.Click here for file


Drosophila. We applied this system to mutations at each of 11 repair loci plus various double mutants and altered dosage genotypes. Most of the mutants were found to suppress one of the pathways with a compensating increase in one or more of the others. Perhaps surprisingly, none of the single mutants suppressed more than one pathway, but they varied widely in how the suppression was compensated. We found several cases in which two or more loci were similar in which pathway was suppressed while differing in how this suppression was compensated. Taken as a whole, the data suggest that the choice of which repair pathway is used for a given DSB occurs by a two-stage “decision circuit” in which the DSB is first placed into one of two pools from which a specific pathway is then selected.The analysis of double-strand break (DSB) repair is complicated by the existence of several pathways utilizing a large number of genes. Moreover, many of these genes have been shown to have multiple roles in DSB repair. To address this complexity we used a repair reporter construct designed to measure multiple repair outcomes simultaneously. This approach provides estimates of the relative usage of several DSB repair pathways in the premeiotic male germline of  DNA is a fragile thread that often breaks. When it does, the cell must find a way to splice the broken ends back together in order to continue its cycle of replication. Cells possess an array of ways to rejoin broken DNA ends, each with advantages and disadvantages. Some are “quick and dirty,” sacrificing accuracy for robustness. They do the basic job of resealing the break but often result in random base changes at the site of the repair. At the other extreme are methods with greater fidelity but added restrictions, such as requiring chromosome replication. We used an experimental system to obtain highly accurate measurements of the relative usage of various repair methods in developing germ cells of fruit flies. The measurements were made in normal flies as well as those carrying mutations at each of 11 genes involved in DNA repair. Most previous studies of these genes focused on specific biochemical pathways. Our results looked at how the repair apparatus as a whole compensates for defects in individual components. The data point to a “decision circuit” for matching each break to a repair method and provide new insight into how our DNA repair machinery protects the genome's integrity. In the last ten years the study of double-strand break (DSB) repair has become more urgent and more difficult. The urgency arises from findings that defects in DSB repair are linked to elevated cancer risks  and phenDrosophila to obtain generalizable results concerning the basic processes. In Drosophila, a large-scale screen for mutagen sensitivity has been useful in identifying DSB repair genes [Drosophila. These include the use of excision of P transposable elements to create breaks [In view of this complexity, it is fortunate that the high degree of conservation in DSB repair systems makes it possible to use model organisms such as ir genes . A variee breaks –23 as wee breaks –27.Drosophila germline [Drosophila version of BLM, the Bloom syndrome gene, is needed to resolve double Holliday junctions via dissolution [The repair reporter construct (Rr3) is designed to yield simultaneous measurements of multiple DSB repair outcomes in the germline . Other sgermline ,30 and ygermline –33. Measgermline –36. Rr3 germline . Anothergermline ,36,37. Ssolution .A major advantage of using Rr3 as opposed to measuring one pathway at a time is that the Rr3 analysis reveals not only which pathway(s) are inhibited by a given condition or genotype, but also which other pathways are used to compensate. This compensation is easily seen as a negative correlation between the relative usage of the various pathways  such thaDsRed, and flanked by a direct repeat of 147 bp  (p = 0.004)   A and in = 0.004) B. The kuctively) A and 3B.lig4 and ku70 mutants, there was no such decrease for the end joining accompanied by long deletions  for cross 1. These deletion events could result when the more typical NHEJ process fails.Despite the pronounced drop in “normal” end-joining repair for eletions C and 3D.lig4 and ku70 mutants was entirely compensated by an increase in SSA, as seen in lig4 were highly significant, and three of the four comparisons for ku70 were also significant. In contrast, there was little or no compensation by HR-h, as shown in DmBlm mutation also present in that genotype. We conclude that SSA provides all or nearly all of the compensation for the loss of NHEJ in lig4 and ku70 mutants. It should be emphasized that this conclusion applies only to breaks within the Rr3 construct where an opportunity for SSA is provided via the 147-bp duplication. The finding that SSA but not HR-h can compensate for the reduction in NHEJ in this class of mutations suggests that there is a pool of DSBs that can “choose” between NHEJ and SSA, but not HR-h. We shall refer to this hypothetical pool of breaks as pool 1 in subsequent discussions.Unexpectedly, we found that the reduction in NHEJ frequencies in DmBlm, rad54, and rad51 as well as in the heterozygotes of rad51. These effects are seen in rad51 homozygotes that reduced long-tract HR-h more than 100-fold, and eliminated short HR-h completely. Null homozygotes of DmBlm and rad54 also reduced both long- and short-tract HR-h  in suppressing SSA is only apparent in experiments 6 and 9 where the endonuclease is supplied from insertion ubiquitin-driven I-SceI endonuclease (UIE)-72C on chromosome 3 and not in experiments 17, 18, or 19 where the endonuclease source is insertion UIE-5B on the X chromosome. As noted previously [mei-9 whose effect on SSA is relatively weak.The effect of eviously , differeDmATR, mus101, and mus301 in both cross 2 , all four of those that suppress HR-h , and three of the four that suppress SSA . No mutation showed a decrease in NHEJΔ frequency. We conclude that NHEJΔ can be increased by a wide range of mutational changes but not reduced. We interpret this finding to indicate that failure of repair via any of the pathways can result in long deletions, but that such deletions are not formed in the normal course of any pathway.We also observed a significant increase in the NHEJΔ outcome for  cross 2 C and for crosses C and 5F.mei-9 mutants was reduced to less than one-fourth the control levels. The other mutants tested, lig4, mus81, DmATR, and mus101, showed no significant difference from the controls. Our interpretation is that mei-9 mutations are defective for some aspect of the repair process in which double Holliday junctions are formed and resolved via strand exchange as opposed to dissolution. The relative rarity of crossing over during DSB repair in Drosophila suggests that this sequence of events is not a major pathway in wild-type flies.We performed further tests on some of the mutants in addition to the Rr3 assays in EX8ku70 deletion shown in ku70 was mutated and to characterize other effects of the mutation. Drosophila Ku70 transgene [EX8ku70 homozygotes had an excess of macrochaetae (large thoracic bristles) that were deformed or reduced in size, and this phenotype was rescued by Ku70 transgenes  mutations [mus101 and mus301  are suppressed and which are used for compensation . By clasuce NHEJ A and 3B d54 loci  was expeutations ,44. On td mus301 A and 5B mus81, although synthetic lethality in the mus81-DmBlm double mutant [mus81 in DSB repair and/or recovery from replication fork collapse in Drosophila. Our finding that no mutation suppressed more than one pathway is perhaps surprising in view of the multiple roles found for many DSB repair genes. This apparent simplicity need not be taken to imply that each gene has a major role in only one pathway, but rather may be interpreted to reflect the robustness of the overall DSB repair system and its ability to compensate for defects in any one component.Of the 11 DSB repair loci in this study, ten of them suppressed exactly one of the three pathways , and none reduced more than one. The only mutation with no differences from wild type in the Rr3 assay was e mutant  suggestslig4 and ku70 mutations are compensated by SSA and not HR-h  showed compensation by NHEJ and HR-h, but the increase in HR-h occurred primarily with long-tract conversion events. One could argue that these eight genes have secondary effects on some of the other outcomes, but it would be necessary to postulate that these secondary effects counterbalance the increase just enough to leave a net result of no significant difference from the control.In the case of premeiotic selection, a testable prediction is that when one type of outcome is knocked down by mutation, all others would increase proportionately. That is, compensation would occur through an increase in all measured outcomes except those directly affected by the mutation. Instead, we found that compensation usually occurred through only one of the alternative outcomes . In factFrom the above observations, we conclude that selection, either post- or premeiotic, is unlikely to account for more than a small proportion of the observed compensation. Instead, we suggest that these compensatory changes reflect the process by which the DSB repair mechanism as a whole channels each break into one of the available pathways.lig4 and ku70 mutants is compensated only by SSA and not HR-h suggests the existence of a pool of DSBs for which SSA and NHEJ are the only options. A second line of evidence for pool 1 is provided by the mutations mus101 and mei-9 in which the reduction in SSA is compensated by an increase in NHEJ but not HR-h , which results in increased usage of the alternative option. For example, the rad51 mutation is hypothesized to prevent most DSBs in pool 2 from being repaired via HR-h, resulting in such breaks being handled by SSA  the frequency was 67% of its heterozygous control (experiments 27 and 28). In contrast, the other two repair outcomes, NHEJ and HR-h, displayed much more severe reductions in the corresponding mutants: lig4 mutants without maternal product produced only 22% as much NHEJ as their controls (experiments 3 and 1), and rad51 homozygotes had less than 1% of the short- or long-tract HR-h frequencies as their controls (experiments 29 and 22). These observations provide a good fit to the model, although other explanations are possible.A second prediction is that mutants with reduced SSA will show a milder degree of reduction compared to those that reduce the other two outcomes. This is because SSA outcomes are drawn from both pools of breaks whereas NHEJ and HR-h each draw DSBs from only a single pool. Consistent with this prediction, we found that in the most severe restriction of SSA  and could be spurious. In addition, we do not see any effect on SSA or NHEJ in cross 1 for DmBlm or top3α even though they are postulated to act at the same segment of the decision circuit as rad51  would be less apparent.Cross 1 can also be represented by a decision circuit diagram, as in  cross 2 C while i cross 2 E and 4H. circuit D. The reas rad51 C. The rerad54 increased usage of SSA for duplications of moderate length, as in At minimum, the diagrams in DmBlm, rad51, and rad54  is not available. This restriction could result from the timing of the break within the cell cycle, as recently reviewed . Thus, prad54 restricts entry into pool 2 suggesting it has an early role in the decision process, acting at or prior to the resection stage. This example serves to emphasize the distinction between the decision process visualized in rad54 appears to act early, versus the biochemical repair process itself where there is evidence that rad54 has one or more later roles [Pool 2 consists of DSBs that are slated to be repaired by conversion (HR-h and HR-s) or SSA, both of which have extensive 5′ resection of the broken ends as a prerequisite ,47. Therer roles ,49.The apparatus for repairing double-strand breaks is ancient and essential. Without this system, the onslaught of DSBs from replication fork collapse, oxidative damage, ambient radiation, and other sources would severely limit any cell lineage. Moreover, the weakening of genomic integrity would make multicellular organisms more susceptible to cancer  and probCrossing and scoring procedures were as described previously ,34–36 exAll DSB repair measurements pertain to the premeiotic germline of the test males whose progeny were scored. These males were always mated within two weeks of when they eclosed to avoid differences related to male age .lig4 and ku70. Details of the procedure and the resulting deletion alleles are in We mobilized nearby P element insertions to generate flanking deletions in the areas of Each estimate of a DSB repair outcome frequency reported in Figure S1(118 KB PDF)Click here for additional data file.Figure S2(91 KB PDF)Click here for additional data file.Figure S3(137 KB PDF)Click here for additional data file.Figure S4(141 KB PDF)Click here for additional data file.Figure S5(87 KB PDF)Click here for additional data file.


KCNJ11 and ABCC8 genes, encoding the Kir6.2 and SUR1 subunit of the pancreatic KATP channel involved in regulation of insulin secretion, account for one third to half of the PNDM cases. Molecular analysis of chromosome 6 anomalies (found in more than 60% in TNDM), and the KCNJ11 and ABCC8 genes encoding Kir6.2 and SUR1, provides a tool to identify TNDM from PNDM in the neonatal period. This analysis also has potentially important therapeutic consequences leading to transfer some patients, those with mutations in KCNJ11 and ABCC8 genes, from insulin therapy to sulfonylureas. Recurrent diabetes is common in patients with "transient" neonatal diabetes mellitus and, consequently, prolonged follow-up is imperative. Realizing how difficult it is to take care of a child of this age with diabetes mellitus should prompt clinicians to transfer these children to specialized centers. Insulin therapy and high caloric intake are the basis of the treatment. Insulin pump may offer an interesting therapeutic tool in this age group in experienced hands.Transient (TNDM) and Permanent (PNDM) Neonatal Diabetes Mellitus are rare conditions occurring in 1:300,000–400,000 live births. TNDM infants develop diabetes in the first few weeks of life but go into remission in a few months, with possible relapse to a permanent diabetes state usually around adolescence or as adults. The pancreatic dysfunction in this condition may be maintained throughout life, with relapse initiated at times of metabolic stress such as puberty or pregnancy. In PNDM, insulin secretory failure occurs in the late fetal or early post-natal period and does not go into remission. Patients with TNDM are more likely to have intrauterine growth retardation and less likely to develop ketoacidosis than patients with PNDM. In TNDM, patients are younger at the diagnosis of diabetes and have lower initial insulin requirements. Considerable overlap occurs between the two groups, so that TNDM cannot be distinguished from PNDM based on clinical features. Very early onset diabetes mellitus seems to be unrelated to autoimmunity in most instances. A number of conditions are associated with PNDM, some of which have been elucidated at the molecular level. Among these, the very recently elucidated mutations in the  Neonatal diabetes mellitus (NDM) is a rare  but potentially devastating metabolic disorder characterized by hyperglycemia combined with low levels of insulin. Two main groups have been recognized on clinical grounds, transient NDM (TNDM) and permanent NDM (PNDM), which differ in the duration of insulin dependence early in the disease. TNDM is a developmental disorder of insulin production that resolves postnatally and represents 50% to 60% of cases of neonatal diabetes. There are no clinical features that can predict whether a neonate with diabetes (but no other dysmorphic features) will eventually have permanent or transient disease. Recently, advances have been made in the understanding of the molecular mechanisms of pancreatic development that are relevant to PNDM and TNDM .Permanent neonatal diabetes mellitus is less common than the transient form of the condition. By definition, diabetes develops in the neonatal period and does not go into remission. There are no clinical features that can predict whether a neonate with diabetes but no other dysmorphic features will eventually have permanent or transient disease, although cases with the permanent form do not always have IUGR as is universally seen in the transient 6q phenotype (see below) Table  6,7]. D. D7]. DiLOT1, PLAGL-1) that regulates cell cycle arrest and apoptosis and also the Pituitary Adenylate Cyclase Activating Polypeptide Receptor 1 (PACAP1) being a potent insulin secretagogue, and the other is the HYMAI gene, whose function is unknown [TNDM is usually sporadic, but paternal transmission has been documented in about one-third of reported patients, some of whom had non-diabetic fathers ,12. Pate unknown . No othe unknown . Paterna unknown . NeverthKCNJ11 encoding the Kir6.2 subunit of the pancreatic ATP-sensitive potassium channels (KATP) couple cell metabolism to electrical activity by regulating potassium movement across the membrane. These channels are made up of an octameric complex with two kind of subunits: four regulatory sulfonylurea receptors (SUR) embracing four pore forming inwardly rectifying potassium channels (Kir). A 1:1 SUR1:KIR6.2 stochiometry is both necessary and sufficient for assembly of active KATP channels. SUR, a member of the ABC transporter family, originates from two separate genes and therefore occurs in several spliced isoforms. SUR1 is found in the pancreatic β-cells and neurons, whereas SUR2A is in heart cells and SUR2B in smooth muscle. The Kir6.2 subunit forms the channel pore in the majority of tissues such as pancreatic β-cells, brain, heart and skeletal muscles, while Kir6.1 can be found in smooth vascular muscle and astrocytes. These different channel forms have different pore properties and adenine nucleotide sensitivity.Mutations in et al. reported in an ethnically diverse patient cohort that six heterozygous activating mutations in the KCNJ11 gene encoding the Kir6.2 subunit of the pancreatic β-cell potassium ATP (KATP) channel caused PNDM in ten probands [KCNJ11 gene for mutations in patients with PNDM recruited through the French network for the study of neonatal diabetes. Seventeen at-term babies with a median age of 64 days at diagnosis of diabetes (range 1–260) were included [et al.; the four novel mutations resulted in amino acid changes of Kir6.2 at positions F35L, G53N, E322K, Y330C. More patients with a Kir6.2 mutation (6/9) were reported to have a smaller birth weight than those without the mutation (2/8), confirming the findings of Gloyn et al. [in utero insulin secretory insufficiency and growth retardation. In conclusion, Kir6.2 mutations are a common cause, from one third to a half of PNDM in Caucasians. This work has opened new avenues for the work-up and treatment of those patients (see below) [Recently, Gloyn probands . Some ofprobands . We screincluded . We idenn et al. ,19. These below) .ATP) channel (composed of SUR1 and KIR6.2 proteins) is a key regulator of insulin release. It is inhibited by the binding of adenine nucleotides to KIR6.2 leading to closing of the channel, and activated by nucleotide binding and/or hydrolysis on SUR1 leading to opening of the channel. The balance of these opposing actions determines the low open channel probability, P0, which controls the excitability of pancreatic β-cells. We hypothesized that activating mutations in ABCC8 which encodes SUR1 cause neonatal diabetes. We therefore screened the 39 exons of ABCC8 in 34 patients diagnosed with permanent or transient neonatal diabetes (ND) of unknown origin in a case series of 73 ND patients [ATP channels. We identified seven missense mutations in nine patients. Four mutations were familial and showed vertical transmission with neonatal and adult onset diabetes; the remaining mutations were de novo and not found in more than 300 non-diabetic or early onset diabetic subjects of similar genetic background. Mutant channels in intact cells and in physiologic concentrations of MgATP had a markedly higher P0 compared with wild type channels. These over-active channels retained sulfonylurea sensitivity and treatment with sulfonylureas achieved euglycemia. Dominant mutations in ABCC8 account for 12% (9/73) of our ND cases. Diabetes results from a novel mechanism whereby the basal Mg-nucleotide-dependent stimulatory action of SUR1 on the KIR pore is elevated and block by sulfonylureas is preserved [As explained above, the ATP-gated potassium  have not so far been found to be associated with permanent neonatal diabetes. On the contrary, mutations in the SUR1 and Kir6.2 subunit have been found in association with transient and permanent neonatal diabetes. Of note is that mutations in Kir6.2 have been found mostly in association with very early forms of diabetes, usually before 6 months of age, whereas the phenotypic variability of SUR1 mutations is broader ,22. Indeng adult .A number of discrete clinical syndromes have been identified as associated with PNDM.IPF-1 (Pro63fsdelC) [The first patient described was a child with pancreatic agenesis and marked endocrine and exocrine failure. Insulin Promoter Factor-1 appeared to be a good candidate for pancreatic agenesis, given its role as a master control of exocrine and endocrine pancreatic development identified from studies of gene disruption , and lat3fsdelC) .IPF-1 acting as a dominant negative inhibitor of wild type IPF-1 activity [IPF-1 mutations can cause autosomal dominant late onset forms of type 2 diabetes that account for around 6% of a French cohort of multiplex type 2 diabetic families.Furthermore, eight individuals from six generations with early onset diabetes akin to type 2 diabetes have been identified within the extended family. These were identified as heterozygotes for the same mutation, with the mutant truncated isoform of activity . The illglucokinase gene and usually leads to mild hyperglycemia in affected individuals [glucokinase gene; this rendered them completely deficient in glycolytic activity, whilst their apparently consanguineous and mild to moderately glucose intolerant parents were heterozygous for the same mutations [glucokinase mutations both in British and French cohorts  has yielded none suggesting that this is unlikely to be a major cause of PNDM [glucokinase mutations is then warranted.MODY 2 is caused by mutations in the ividuals . Glucokiutations . A searc of PNDM ,30. HoweFOXP3 gene that encodes a forkhead domain-containing protein [Foxp3 is characterized by over-proliferation of CD4+/CD8-T lymphocytes with multi-organ infiltration. The males die 15–25 days after birth [A number of authors have reported an X-linked syndrome with a combination of exfoliative dermatitis, intractable diarrhea with villous atrophy, hemolytic anemia, autoimmune thyroid disease and neonatal onset diabetes. Most children die in the first year of life with overwhelming sepsis . In some protein . The scuer birth . It has et al. used two consanguineous families to map the condition to the locus 2p12 [EIF2AK3 that is highly expressed in islet cells and acts as a regulator of protein synthesis. Proteins and insulin are manufactured in the endoplasmic reticulum (ER). In response to environmental stresses, cells down regulate protein synthesis by phosphorylation of the alpha subunit of eukaryotic translation initiation factor-2  by eukaryotic translation initiation factor-2 kinase3 (EIF2AK3). Mal-folded proteins in the ER inhibit further translation initiation mediated by increased phosphorylation of eif2-alpha. A targeted mutation of the mouse Eif2ak3 gene (PERK) led to an accumulation of mal-folded proteins in the ER, with resultant abnormally elevated protein synthesis and increased stress on ER folding machinery [EIF2AK3 segregating with the disorder in each family [Wolcott-Rallison syndrome is an autosomal recessive disorder characterized by infancy onset  diabetes associated with a spondyloepiphyseal dysplasia. In addition, there is a constellation of other features such as hepatomegaly, mental retardation, renal failure and early death . In 2000cus 2p12 . Within achinery . PERK isachinery . Furtherh family .et al. described two boys with X-linked phosphoribosyl-ATP pyrophosphatase hyperactivity who became diabetic on day one of life. Glucose intolerance persisted throughout life, although there were periods off insulin, as the children grew older. Both boys had other major problems including mental retardation, ataxia and progressive axonal neuropathy. The mother also had hyperuricemia (gout) and glucose intolerance with a history of gestational diabetes [In 1992, Christen diabetes .et al. described a condition of neonatal diabetes associated with severe hypoplasia of the pancreas (only head and uncus present) and congenital cyanotic heart disease in a single family with apparent autosomal dominant inheritance. Not all the cases developed diabetes as a neonate, the timing is probably related to the size of remaining pancreatic tissue [In 1994, Yorifuji c tissue .β-cells and neuronal tissues: dysfunction of any of these might explain the two components of this syndrome [Recently, another severe syndrome was described in which three members of a consanguineous family developed neonatal diabetes and cerebellar hypoplasia. An autosomal recessive inheritance pattern was suggested. The infants all died within a few months of birth from a combination of metabolic dysfunction, respiratory compromise and sepsis . Interessyndrome . Indeed,syndrome . These psyndrome .Very recently, mutations in Glis3 (another transcription factor) were found to explain a syndrome which associated neonatal diabetes, hypothyroidism, congenital glaucoma, kidney cysts and hepatic fibrosis .β-cells targeted autoimmune attack [A single case report has suggested that maternal enterovirus (echovirus 6) infection in pregnancy (end of first trimester) can lead to autoimmune, neonatal onset diabetes with the presence of anti-insulin and glutamic acid decarboxylase antibodies at birth or very soon after birth. In this female child (ruling out IPEX), the pancreas was very hypoplastic and the authors suggested a role for maternally transmitted enterovirus either by direct influence on pancreatic organogenesis or through aggressive e attack .It is worth mentioning that neonatal diabetes may also exist in the context of a mitochondrial disorder . It is uKCNJ11 and ABCC8 genes (encoding Kir6.2 and SUR1 respectively) provide a tool for identifying transient from permanent neonatal diabetes mellitus in the neonatal period; 7) About 50% of the PNDM cases are linked to potassium channel mutation which has potentially important therapeutic consequences leading to transfer some patients from insulin therapy to sulfonylureas.The following conclusions can be drawn from current knowledge on neonatal diabetes mellitus: 1) Patients with TNDM are more likely to have intrauterine growth retardation and less likely to develop ketoacidosis than patients with PNDM; 2) TNDM patients are younger at the age of diagnosis of diabetes and have lower initial insulin requirements; 3) Considerable overlap occurs between the two groups, so that TNDM cannot be distinguished from PNDM based on clinical features; 4) Very early onset diabetes mellitus seems to be unrelated to autoimmunity in most instances; 5) Recurrent diabetes is common in patients with "transient" neonatal diabetes mellitus and, consequently, prolonged follow-up is imperative; 6) Molecular analysis of chromosome 6 anomalies, the The uniparental disomy of the chromosome 6 can be evidenced by the analysis of polymorphic markers present on the chromosome 6; meiotic segregation of the chromosomes can be determined by comparing the allelic profiles of polymorphic markers in the child and his parents. Usually, a total uniparental disomy of the chromosome 6 is evidenced, but partial one can also be found. This is why markers close to the region of interest 6q24) should be chosen. Chromosome 6 duplications can also be evidenced by that technique  in all the cases of neonatal diabetes requiring subcutaneous insulin therapy for more than 15 days. We report here our experience in the five cases where initiation of the CSII was done in our departments. Four of the cases were TNDM (follow-up 7 months to 10 years). The practical aspects of the treatment were as follows. During the first days, the daily dose requirement was evaluated with intravenous insulin and glucose infusions. When good glycemic control was obtained, CSII therapy was started. Insulin was diluted to 4 to 10 units per milliliter. Insulin strategy to start CSII depended on the feeding conditions. Under enteral continuous feeding, 100% of the total daily dose was administered as basal rate. Under bottle feeding, the basal rate represented 30% of the total daily dose and boluses 70%, with the same insulin dose before each meal . Blood glucose was monitored every 3 to 4 hours. Basal rate was adjusted on the night blood glucose measurements and boluses on the post-prandial ones. CSII therapy was started between day 7 and 55, one to 13 days after initiation of insulin therapy. During the first month of CSII therapy (dose at day 15 of CSII: 0.3 to 1 unit per kg/day), good glycemic control was achieved on a mean number of 240 blood glucose measurements (5 patients). Mean blood glucose was 1.73 g/l, no severe hypoglycemia was noted, and the mean number of hypoglycemia episodes (blood glucose <0.6 g/l) was 4.2 per month. Similar excellent results were achieved for the rest of the CSII in both the TNDM and the PNDM cases. We did not observe any cutaneous side effects. We conclude that during the neonatal period, CSII therapy is safe, more physiological, more accurate and easier to manage than injections. CSII allows us to match the insulin requirements of a newborn. CSII requires management and supervision by a experienced team of physicians and nurses.Realizing how difficult it is to take care of a child of this age with diabetes mellitus should prompt clinicians to transfer these children to specialized centers. Insulin therapy and high caloric intake are the basis of the treatment. Insulin pump may offer an interesting therapeutic tool in this age group in experienced hands.The risk of recurrence is different according to the "transient" or permanent form of the disease and to the different molecular mechanisms identified.In the case of uniparental disomy, none of the allele of the mother is found in the proband, the risk of recurrence does not exist in theory and there is no transmission by the child. Partial uniparental disomy have been described, linked to a post-zygotic mechanism. The recurrence risk is probably weak or close to zero, as the anomaly is post-zygotic.de novo mutation, there is a priori no risk of recurrence, except if germinal mosaicism exists (not described so far). Concerning the risk of transmission, fathers will transmit both the genetic defect and the disease. Half of the children will have the disease, which will be present equally in boys and girls. If the mother has the anomaly, her children will not have the disease but the male offspring will eventually pass on the disease.If partial duplication is present in the propositus, there is a risk of disease recurrence in the family and parents have to be tested. Carriers of the duplication have a 50% risk of transmitting the defect. If it is a Imprinting anomaly: the logic says that the mother should pass it on (imprinting relaxation) but so far no familial case is known and the risk of transmission is unknown. Indeed, the cause of the imprinting relaxation is not identified and the identified children with this anomaly are too young to procreate.EIF2AK, Glis3, PTF1A, and PDX1 genes). IPEX syndrome is an X linked disorder.Recurrence risk is 25% in the recessive autosomal disorders (The mutation in the genes encoding the potassium channel subunits are transmitted in the heterozygous state in a dominant way. Moreover, transmission by germinal mosaicism has here been described.In the neonatal period, the prognosis is linked to the severity of the disease, the degree of dehydration and acidosis, as well the rapidity with which the disease is recognized and treated. In the following period, the prognosis is determined by the associated malformations and lesions. For example, in the case of potassium channel anomalies, neuropsychological and neuromuscular disturbances can be present. Finally, the prognosis rely on the metabolic control, as in all the forms of diabetes mellitus, which will determine the timing of appearance of the long standing diabetes complications.IPF-1 mutation is important in MODY 4 and in some familial forms of early onset type 2 diabetes and the SUR1 mutation plays a role in newborn diabetes as well as in type 2 diabetes in young adults. We hope that elucidating the etiology of other forms of neonatal diabetes will provide information on normal pancreatic development and the basis of the pathology underlying pancreatic dysfunction.Neonatal diabetes is a rare condition. However, it is probably of great relevance to our understanding of the cause of type 2 diabetes within the general population. We believe these rare single gene disorders are natural models for identifying new genes with possible relevance to type 2 diabetes. As discussed, the


The results indicate that ULO effects on E2 concentrations are asymmetric, vary during the estrous cycle, and partially depend on the cholinergic innervation.There is evidence that one gonad has functional predominance. The present study analyzed the acute effects of unilateral ovariectomy (ULO) and blocking the cholinergic system, by injecting atropine sulfate (ATR), on estradiol  resulted in significantly higher E2 concentrations than animals with ULO of the left ovary (right ovary in situ). ATR treatment to ULO rats on D1 resulted in a significant drop of E2 serum concentrations. ULO rats treated with ATR on D2 or P, resulted in an asymmetrical E2 secretion response; when the right ovary remained in situ an increase in E2 was observed, and a decrease when the left ovary remained in situ.ULO of the right ovary (left ovary 2 from the missing ovary is different and varies during the estrous cycle. The results also suggest that the cholinergic system participates in regulating ovarian E2 secretion. Such participation varies according to the ovary remaining in situ and the stage of the estrous cycle of the animal.The results obtained in the present study suggest that each ovary's ability to compensate the secretion of E2 secretion, and also supports the idea of cross-talk between the ovaries, via a neural communication, that modulates E2 secretion.The results agree with previously stated hypothesis of a neural pathway arising from the peritoneum that participates in regulating E Estradiol secretion is regulated by pituitary . The effects of these hormones are modulated by neurotransmitters released by the intrinsic ovarian innervation near the follicular wall. Acetylcholine produced by the follicle may be one of the neurotransmitters participating in modulating the effects of pituitary hormones on the follicle -3.in situ 2 [4 [10]2 [4 . In the 2 [4 . Since pA study analyzing the distribution of sensory neurons innervating the peritoneum showed that when tracer was placed on the area where the peritoneum covers the abdominal wall, labeled neurons were observed only in the ipsilateral dorsal root ganglia . The autAccording to Stener-Victorin et al. 19) repeated electro-acupunture treatments in rats with polycystic ovary syndrome (PCO), induced by a single injection of estradiol valerate, resulted in lower nerve growth factor (NGF) concentrations at the ovarian level than in non-electro-acupunture treated PCO rats. In our experiments, perforating the peritoneum affected the same somatic segments employed by Stener-Victorin et. al. [ repeated2 serum concentrations did not increase after left or bilateral perforation of the peritoneum, we suppose that the neural information originating from the peritoneum inhibits the mechanisms regulating aromatase activity within the follicle.We presume that peritoneum surgery resulted in an increase of NGF concentrations at the ovarian level, which in turn induced hyper-androgenism, as observed in women with PCO . Previou4 and E2 concentrations in the ovarian vein effluent [2 concentrations [2 concentrations.One of the ovaries' sources of catecholamines arrives through the SON. In the ovary, the SON fibers are mainly distributed in the peri-follicular theca layer, and in close relation with the cells of the theca interna ,22. Secteffluent , while ttrations . Therefo2 concentration in the blood. Present results indicate that injecting ATR, before unilateral or bilateral perforation of the peritoneum, to rats in D1 or P, results in lower E2serum concentrations; leading us to think that some of the neural fibers present in the peritoneum are muscarinic. It is also possible that blocking the cholinergic innervation, by ATR treatment, results in lower adrenaline and norepinephrine release by the adrenal medulla.According to Erdö, et. al.  and Lasz2 secretion inhibitory mechanism, that the sensory pathway arising from the right side has a stimulatory effect, and that both are mediated by the cholinergic muscarinic system.Our results, and those of others, suggest that stimulating on D2 the sensory receptors located on the left side of the peritoneum triggers an E4 serum concentrations originating from the adrenals [4 and androgens are precursors in the synthesis of E2, we presume that this mechanism may explain the increase in E2 serum concentrations observed in rats with peritoneum perforation previously injected with ATR.Previously, we showed that injecting ATR to rats in D2 results in increases of Padrenals , withoutadrenals . Since P2 secretion regulation during the estrous cycle is that the effects of the cholinergic system take place through changes at the celiac ganglion level. According to Aguado and Ojeda [4 secretion in the celiac ganglion-SON-ovary preparation obtained from rats in D1 or D2, while the preparation obtained from rats in P resulted in only a moderate stimulation. Since there is evidence that fibers from the vagus nerve innervate neurons in the celiac ganglion [Another possibility explaining the differences on End Ojeda , acetylcganglion , we pres2 serum concentrations in rats with ULO, present results suggest that the capacity to release E2 by the left and right ovaries varies during the estrous cycle. We presume that the left ovary releases more E2 than the right one. As previously proposed, another possibility is that neural communication between the ovaries modulates E2 secretion.Based on the differences in E