HTR1A a Novel Type 1 Diabetes Susceptibility Gene on Chromosome 5p13-q13

Background: We have previously performed a genome-wide linkage study in Scandinavian Type 1 diabetes (T1D) families. In the Swedish families, we detected suggestive linkage (LOD less than= 2.2) to the chromosome 5p13-q13 region. The aim of our study was to investigate the linked region in search for possible T1D susceptibility genes. Methodology/Principal Findings: Microsatellites were genotyped in the Scandinavian families to fine-map the previously linked region. Further, SNPs were genotyped in Swedish and Danish families as well as Swedish sporadic cases. In the Swedish families we detected genome-wide significant linkage to the 5-hydroxytryptamine receptor 1A (HTR1A) gene (LOD 3.98, pless than9.8x10(-6)). Markers tagging two separate genes; the ring finger protein 180 (RNF180) and HTR1A showed association to T1D in the Swedish and Danish families (pless than0.002, pless than0.001 respectively). The association was not confirmed in sporadic cases. Conditional analysis indicates that the primary association was to HTR1A. Quantitative PCR show that transcripts of both HTR1A and RNF180 are present in human islets of Langerhans. Moreover, immunohistochemical analysis confirmed the presence of the 5-HTR1A protein in isolated human islets of Langerhans as well as in sections of human pancreas. Conclusions: We have identified and confirmed the association of both HTR1A and RFN180, two genes in high linkage disequilibrium (LD) to T1D in two separate family materials. As both HTR1A and RFN180 were expressed at the mRNA level and HTR1A as protein in human islets of Langerhans, we suggest that HTR1A may affect T1D susceptibility by modulating the initial autoimmune attack or either islet regeneration, insulin release, or both

Synthesis of Novel Inhibitors of IdeS, a Bacterial Cysteine Protease Including Studies of Stereoselective Reductive Aminations

Abstract The cysteine protease IdeS is an IgG degrading enzyme secreted by the bacterium Streptococcus pyogenes to evade the human immune system. In this thesis several inhibitors of IdeS have been synthesized and evaluated. Such inhibitors should be highly useful when elucidating the detailed mechanism of IdeS action. They might also have a potential as treatment of acute and severe infections caused by the bacteria. Further, IdeS has a therapeutic application of its own due to the proteolytic ability and an IdeS inhibitor might contribute during the development. Only irreversible, unselective inhibitors of IdeS were known five years ago. In this thesis, three strategies with the aim to synthesize and identify more inhibitors have been undertaken. Focus was first set on compounds with a substructure resembling the known inhibitors but with reversible warheads, i.e. nitrile, azide and aldehyde functions. The aldehyde derivatives were found to provide the first reversible inhibitors of IdeS. Then, to avoid covalent interactions and obtain more selective inhibitors, a substrate based strategy was undertaken. A 3-aminopiperidine fragment was used as replacement of either of the two residues adjacent to the scissile bond in IgG. Such fragments can be synthesized from N-protected 3-aminopiperidone and amino acid esters in reductive aminations in which a stereogenic center is formed. A series of di-, tri- and tetrapeptide analogues, together with eight peptides covering the cleavage site of IgG, were screened for their capacity to inhibit the cysteine proteases IdeS, SpeB and papain. Several analogues showed inhibition capacity, two compounds showed also high selectivity for IdeS. In contrast, none of the tested peptides showed any inhibition. Computational docking studies indicate that the identified IdeS peptide analogues and the non-active peptides do not share the same binding site in IdeS. Probably, the piperidine moiety hinders the inhibitor to enter the catalytic site. A more detailed study of the stereoselectivity in the reductive aminations affording the 3-aminopiperidine fragment showed that a large protecting group (trityl) together with a large reducing agent (NaBH(O-2- ethylhexanoyl)3) gave the highest diastereomeric ratio. The highest ratio obtained was 21:79 when Lproline methyl ester was used. The newly formed stereogenic center had the R-configuration, determined by chemical correlation. Computer based conformational analysis combined with Boltzmann distribution calculations implies an axial attack by the reducing reagent on the intermediary imine. To improve the potency of the two identified di- and tripeptide analogues synthetic routes to conformationally restricted N-containing bicyclic derivatives was undertaken in a third strategy. Five compounds with different bicyclic scaffolds were screened for their inhibition capacity towards IdeS and papain. One of the compounds was able to inhibit the first step of proteolytic cleavage of IgG by IdeS, a process usually completed in seconds

Oral cladribine for B-cell chronic lymphocytic leukaemia: Report of a phase II trial with a 3-d, 3-weekly schedule in untreated and pretreated patients, and a long-term follow-up of 126 previously untreated patients

A phase II study was undertaken to evaluate the efficacy and toxicity of a new schedule of cladribine administration (10 mg/m(2) orally daily for 3 d every 3 weeks) in 107 patients with B-cell chronic lymphocytic leukaemia (CLL). To minimize toxicity. treatment withdrawal criteria were defined. The results of the h 3 previously untreated patients were retrospectively compared with 0 3 from an earlier study using a 5-d monthly, schedule. The compiled data were analysed for prognostic factors for survival. No significant difference regarding response were seen in the two cohorts of the 126 previously untreated patients. The complete response (CR), nodular partial response (nPR) and partial response (PR) rates were 15% 21% and 41%. Quality of response had no impact on survival. The 3- and 5-year overall survival for previously untreated patients was 73% and 58%, respectively, with a median follow-up of 54 months, Pretreatment haemoglobin < 11.0 g/dl and elevated beta-2-microglobulin had a negative influence oil Survival. Major infections occurred in 21% of patients in the 3-d study compared with 35% in the 5-d study. The overall response (OR) and CR rates in the 40 previously treated patients were 34% and 5% respectively, Median overall survival was 24 months and median progression-free survival for responding patients was 14 months. Cladribine used as a single agent is an effective treatment with an acceptable safety profile for pretreated and untreated B-CLL. The achievement of complete remission was not it prerequisite for long-term survival

3-Aminopiperidine-Based Peptide Analogues as the First Selective Noncovalent Inhibitors of the Bacterial Cysteine Protease IdeS

A series of eight peptides corresponding to the amino acid sequence of the hinge region of IgG and 17 newly synthesized peptide analogues containing a piperidine moiety as a replacement of a glycine residue were tested as potential inhibitors of the bacterial IgG degrading enzyme of Streptococcus pyogenes, IdeS. None of the peptides showed any inhibitory activity of IdeS, but several piperidine-based analogues were identified as inhibitors. Two different analysis methods were used: an SDS-PAGE based assay to detect IgG cleavage products and a surface plasmon resonance spectroscopy based assay to quantify the degree of inhibition. To investigate the selectivity of the inhibitors for IdeS, all compounds were screened against two other related cysteine proteases (SpeB and papain). The selectivity results show that larger analogues that are active inhibitors of IdeS are even more potent as inhibitors of papain, whereas smaller analogues that are active inhibitors of IdeS inhibit neither SpeB nor papain. Two compounds were identified that exhibit high selectivity against IdeS and will be used for further studies

Association to the chromosome 5p13 region in Danish multiplex/simplex families, BDD cohort and the Swedish case-control material.

<p>Allele frequencies and p-values for the Danish multiplex-simplex families and p-values for the Swedish cases-control and BDD studies were obtained from Unphased v. 3.0.6. P-values for the BDD cohort were obtained using controls from the Swedish MS case-control study.</p><p>Genotype frequencies and p-values for the genotype test were obtained by using chi2 test.</p

Association to the chromosome 5p13 region in Swedish multiplex/simplex families.

<p>SNP location (bp) was obtained from “HapMap Data Rel 19/phasell Oct 05, on NCBI B34 assembly, dbSNP b124” in HapMap.</p><p>Minor allele frequencies and p-values for the Swedish multiple/simplex families were obtained from Unphased v. 3.0.6. Hardy-Weinberg (H–W) values were calculated amongst controls.</p

a–b. Linkage and association analysis in Scandinavian families and case-control cohort.

<p>Linkage analysis of T1D on chromsome 5 in Swedish, -Danish and -Norwegian multiplex families (a). The dashed line represents multipoint linkage from the original scan in the Swedish, Danish and Norwegian families <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035439#pone.0035439-Nerup1" target="_blank">[4]</a>. The thin dark grey line represents the “fine mapping” including 36 microsatellites in all Scandinavian families. A region between D5S407 and D5S428 (at approx 41.5 cM in figure) showed a linkage of LOD 2.16. The dotted line represents the Swedish families using all 40 microsattelites (multipoint). Here, D5S2000 showed strongest linkage (LOD 2.70). The black diamonds represent the “fine mapping” single point analysis in the Swedish families. In the singlepoint analysis for the Swedish families, D5S2048 showed strong linkage (LOD 2.97). The black thick line represents linkage in Swedish families to T1D on chromosome using an extra 4 microsattelites and 61 SNPs, reveiling three linked peaks where the most strongly linked region was the 5p13-q13 (at approx 43 cM on figure) region (LOD 2.7 for rs6295). In the singlepoint analysis using all 61 SNPs and 4 extra microsattelites (white diamonds) rs878567 and rs6295 showed genome-wide significant linkage (LOD 3.9 and LOD 3.65 respectively). Linkage was calculated using the Exponential Equal Weighting model in the Allegro program.SNP association for the Swedish sporadic cases and controls and Swedish and Danish multiplex families (b) was calculated from rs1158292 (63 001 317 bp) to rs6880454 (63 5028 02 bp). Swedish families (diamonds). Danish families (squares), DISS2 (triangles) and BDD (crosses). Association analysis was carried out in the Unphased program. For the BDD cohort, association was calculated using controls included in the Swedish (MS) EIMS study.</p