Mucopolysaccharidosis type IVA: common double deletion in the N-acetylgalactosamine-6-sulfatase gene (GALNS)

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by a deficiency in N-acetylgalactosamine-6-sulfatase (GALNS). We found two separate deletions of nearly 8.0 and 6.0 kb in the GALNS gene, including some exons. There are Alu repetitive elements near the breakpoints of the 8.0-kb deletion, and this deletion resulted from an Alu—Alu recombination. The other 6.0-kb deletion involved illegitimate recombinational events between incomplete short direct repeats of 8 bp at deletion breakpoints. The same rearrangement has been observed in a heteroallelic state in four unrelated patients. This is the first documentation of a common double deletion a gene that is not a member of a gene cluster.     

Computational analysis of human N-acetylgalactosamine-6-sulfate sulfatase enzyme: an update in genotype–phenotype correlation for Morquio A

Mucopolysaccharidosis IV A (MPS IV A) is a lysosomal storage disease produced by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) enzyme. Although genotype–phenotype correlations have been reported, these approaches have not enabled to establish a complete genotype–phenotype correlation, and they have not considered a ligand–enzyme interaction. In this study, we expanded the in silico evaluation of GALNS mutations by using several bioinformatics tools. Tertiary GALNS structure was modeled and used for molecular docking against galactose-6-sulfate, N-acetylgalactosamine-6-sulfate, keratan sulfate, chondroitin-6-sulfate, and the artificial substrate 4-methylumbelliferyl-β-d-galactopyranoside-6-sulfate. Furthermore, we considered the evolutionary residue conservation, change conservativeness, position within GALNS structure, and the impact of amino acid substitution on the structure and function of GALNS. Molecular docking showed that amino acids involved in ligand interaction correlated with those observed in other human sulfatases, and mutations within the active cavity reduced affinity of all evaluated ligands. Combination of several bioinformatics approaches allowed to explaine 90 % of the missense mutations affecting GALNS, and the prediction of the phenotype for another 21 missense mutations. In summary, we have shown for the first time a docking evaluation of natural and artificial ligands for human GALNS, and proposed an update in genotype–phenotype correlation for Morquio A, based on the use of multiple parameters to predict the disease severity.     

Enzyme Replacement in a Human Model of Mucopolysaccharidosis IVA In Vitro and Its Biodistribution in the Cartilage of Wild Type Mice

Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is a lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), an enzyme that degrades keratan sulfate (KS). Currently no therapy for MPS IVA is available. We produced recombinant human (rh)GALNS as a potential enzyme replacement therapy for MPS IVA. Chinese hamster ovary cells stably overexpressing GALNS and sulfatase modifying factor-1 were used to produce active (∼2 U/mg) and pure (≥97%) rhGALNS. The recombinant enzyme was phosphorylated and was dose-dependently taken up by mannose-6-phosphate receptor (K_uptake = 2.5 nM), thereby restoring enzyme activity in MPS IVA fibroblasts. In the absence of an animal model with a skeletal phenotype, we established chondrocytes isolated from two MPS IVA patients as a disease model in vitro. MPS IVA chondrocyte GALNS activity was not detectable and the cells exhibited KS storage up to 11-fold higher than unaffected chondrocytes. MPS IVA chondrocytes internalized rhGALNS into lysosomes, resulting in normalization of enzyme activity and decrease in KS storage. rhGALNS treatment also modulated gene expression, increasing expression of chondrogenic genes Collagen II, Collagen X, Aggrecan and Sox9 and decreasing abnormal expression of Collagen I. Intravenous administration of rhGALNS resulted in biodistribution throughout all layers of the heart valve and the entire thickness of the growth plate in wild-type mice. We show that enzyme replacement therapy with recombinant human GALNS results in clearance of keratan sulfate accumulation, and that such treatment ameliorates aberrant gene expression in human chondrocytes in vitro. Penetration of the therapeutic enzyme throughout poorly vascularized, but clinically relevant tissues, including growth plate cartilage and heart valve, as well as macrophages and hepatocytes in wild-type mouse, further supports development of rhGALNS as enzyme replacement therapy for MPS IVA.     

Negative association between asthma and variants of CC16(CC10) on chromosome 11q13 in British and Japanese populations

The gene encoding Clara cell-derived inflammatory molecule CC16 has been cited as a candidate gene for atopic asthma on chromosome 11q13. A genetic association study was performed with variants of the CC16 gene on chromosome 11q13 in relation to asthma in British (n=275) and Japanese (n=300) populations. No significant association was found between asthma and CC16 genotypes, irrespective of atopic status in these two populations. These data suggest that CC16 might not be the major locus for asthma on 11q13. Received: 22 December 1997 / Accepted: 9 February 1998     

Assignment of anonymous DNA probes to specific intervals of human chromosomes 16 and X

Summary Anonymous DNA probes mapping to human chromosome 16 and the distal region of the human X chromosome were isolated from a genomic library constructed using lambda EMBL3 and DNA from a mouse/human hybrid. The hybrid cell contained a der(16)t(X;16)(q26;q24) as the only human chromosome. Fifty clones were isolated using total human DNA as a hybridisation probe. Forty six clones contained single copy DNA in addition to the repetitive DNA. Pre-reassociation with sonicated human DNA was used to map these clones by a combination of Southern blot analysis of a hybrid cell panel containing fragments of chromosomes 16 and X and in situ hybridisation. One clone mapped to 16pter 16p13.11, one clone to 16p13.316p13.11, four clones to 16p13.316p13.13, two clones to 16p13.1316p13.11, one clone to 16p13.11, seven clones to 16p13.1116q12 or 16q13, four clones to 16q12 or 16q13, three clones to 16q1316q22.1, four clones to 16q22.10516q24, and nineteen clones to Xq26Xqter. Two clones mapping to 16p13 detected RFLPs. VK5 (D16S94) detected an MspI RFLP, PIC 0.37. VK20 (D16S96) detected a TaqI RFLP, PIC 0.37 and two MspI RFLPs, PIC 0.30 and 0.50. The adult polycystic kidney disease locus (PKD1) has also been assigned to 16p13. The RFLPs described will be of use for genetic counselling and in the isolation of the PKD1 gene. Similarly, the X clones may be used to isolate RFLPs for genetic counselling and the isolation of genes for the many diseases that map to Xq26qter.     

On the appropriateness of use of a continuous formulation for the modelling of discrete multireactant systems following Michaëlis–Menten kinetics

The possibility of solving the mass balances to a multiplicity of substrates within a CSTR in the presence of a chemical reaction following Michaelis-Menten kinetics using the assumption that the discrete distribution of said substrates is well approximated by an equivalent continuous distribution on the molecular weight is explored. The applicability of such reasoning is tested with a convenient numerical example. In addition to providing the limiting behavior of the discrete formulation as the number of homologous substrates increases, the continuous formulation yields in general simpler functional forms for the final distribution of substrates than the discrete counterpart due to the recursive nature of the solution in the latter case.List of Symbols C{N. M} mol/m³ concentration of substrate containing N monomer residues each with molecular weight M - {N, M} normalized value of C{N. M} - C {M} mol/m³ da concentration of substrate of molecular weight M - _in normalized value of C {M} at the i-th iteration of a finite difference method - {M} normalized value of C {M} - C ₀{N.M} mol/m³ inlet concentration of substrate containing N monomer residues each with molecular weight M - {N ·M} normalized value of C₀{N. M} - _{0 i} normalized value of C ₀ {M} at the i-th iteration of a finite difference method - C ₀ {M} mol/m³ da initial concentration of substrate of molecular weight M - C _tot mol/m³ (constant) overall concentration of substrates (discrete model) - C _tot mol/m³ (constant) overall concentration of substrates (continuous model) - D deviation of the continuous approach relative to the discrete approach - i dummy integer variable - I arbitrary integration constant - j dummy integer variable - k dummy integer variable - K _m mol/m³ Michaëlis-Menten constant for the substrates - l dummy integer variable - M da molecular weight of substrate - M normalized value of M - M da maximum molecular weight of a reacting substrate - N number of monomer residues of a reacting substrate - N maximum number of monomer residues of a reacting substrate - N total number of increments for the finite difference method - Q m³/s volumetric flow rate of liquid through the reactor - S inert product molecule - S _i substrate containing i monomer residues - V m³ volume of the reactor - v _max mol/m³ s reaction rate under saturating conditions of the enzyme active site with substrate - v _max{N. M} mol/m³ s reaction rate under saturating conditions of the enzyme active site with substrate containing N monomer residues with molecular weight M - _max{N · M} dimensionless value of v_max{N. M} (discrete model) - _max{M} dimensionless value of v _max {M} (continuous model) - mol/m³ s molecular weight-averaged value of v_max (discrete model) - mol.da/m³s molecular weight-averaged value of v_max (continuous model) - v _max {M} mol.da/m³s reaction rate under saturating conditions of the enzyme active site with substrate with molecular weight M - _max {M} dimensionless value of v_max{M} - _{max, (i)} dimensionless value of v_max{M} at the i-th iteration of a finite difference method - v _max mol/m³ s reference constant value of v _max Greek Symbols dimensionless operating parameter (discrete distribution) - dimensionless operating parameter (continuous distribution) - M da (average) molecular weight of a monomeric subunit - M selected increment for the finite difference method - auxiliary corrective factor (discrete model)     

The Structure of Human GALNS Reveals the Molecular Basis for Mucopolysaccharidosis IV A

Lysosomal enzymes catalyze the breakdown of macromolecules in the cell. In humans, loss of activity of a lysosomal enzyme leads to an inherited metabolic defect known as a lysosomal storage disorder. The human lysosomal enzyme galactosamine-6-sulfatase (GALNS, also known as N-acetylgalactosamine-6-sulfatase and GalN6S; E.C. 3.1.6.4) is deficient in patients with the lysosomal storage disease mucopolysaccharidosis IV A (also known as MPS IV A and Morquio A). Here, we report the three-dimensional structure of human GALNS, determined by X-ray crystallography at 2.2 Å resolution. The structure reveals a catalytic gem diol nucleophile derived from modification of a cysteine side chain. The active site of GALNS is a large, positively charged trench suitable for binding polyanionic substrates such as keratan sulfate and chondroitin-6-sulfate. Enzymatic assays on the insect‐cell-expressed human GALNS indicate activity against synthetic substrates and inhibition by both substrate and product. Mapping 120 MPS IV A missense mutations onto the structure reveals that a majority of mutations affect the hydrophobic core of the structure, indicating that most MPS IV A cases result from misfolding of GALNS. Comparison of the structure of GALNS to paralogous sulfatases shows a wide variety of active‐site geometries in the family but strict conservation of the catalytic machinery. Overall, the structure and the known mutations establish the molecular basis for MPS IV A and for the larger MPS family of diseases.     

Dense‐map genome scan for dyslexia supports loci at 4q13, 16p12, 17q22; suggests novel locus at 7q36

Analysis of genetic linkage to dyslexia was performed using 133,165 array‐based SNPs genotyped in 718 persons from 101 dyslexia‐affected families. Results showed five linkage peaks with lod scores >2.3 (4q13.1, 7q36.1‐q36.2, 7q36.3, 16p12.1, and 17q22). Of these five regions, three have been previously implicated in dyslexia (4q13.1, 16p12.1, and 17q22), three have been implicated in attention‐deficit hyperactivity disorder (ADHD, which highly co‐occurs with dyslexia; 4q13.1, 7q36.3, 16p12.1) and four have been implicated in autism (a condition characterized by language deficits; 7q36.1‐q36.2, 7q36.3, 16p12.1, and 17q22). These results highlight the reproducibility of dyslexia linkage signals, even without formally significant lod scores, and suggest dyslexia predisposing genes with relatively major effects and locus heterogeneity. The largest lod score (2.80) occurred at 17q22 within the MSI2 gene, involved in neuronal stem cell lineage proliferation. Interestingly, the 4q13.1 linkage peak (lod 2.34) occurred immediately upstream of the LPHN3 gene, recently reported both linked and associated with ADHD. Separate analyses of larger pedigrees revealed lods >2.3 at 1–3 regions per family; one family showed strong linkage (lod 2.9) to a known dyslexia locus (18p11) not detected in our overall data, demonstrating the value of analyzing single large pedigrees. Association analysis identified no SNPs with genome‐wide significance, although a borderline significant SNP (P = 6 × 10^–7) occurred at 5q35.1 near FGF18, involved in laminar positioning of cortical neurons during development. We conclude that dyslexia genes with relatively major effects exist, are detectable by linkage analysis despite genetic heterogeneity, and show substantial overlapping predisposition with ADHD and autism.     

A linkage group with FRA16B (the fragile site at 16q22.1)

Summary Polymorphic DNA markers located in bands 16q13, 16q21 and 16q22 were examined for recombination with FRA16B, the fragile site at 16q22.100. A tight linkage cluster D16S10-FRA16B-D16S4-HP was established. There were no recombinants between D16S10 and D16S4, which flank FRA16B. The markers D16S10 and D16S4 are in close proximity on the genetic map and delineate a small chromosomal segment, which contains the distamycin A-inducible fragile site.     

Enzyme replacement therapy for Morquio A: an active recombinant <Emphasis Type="Italic">N</Emphasis>-acetylgalactosamine-6-sulfate sulfatase produced in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency. Currently no effective therapies exist for MPS IVA. In this work, production of a recombinant GALNS enzyme (rGALNS) in Escherichia coli BL21 strain was studied. At shake scale, the effect of glucose concentration on microorganism growth, and microorganism culture and induction times on rGALNS production were evaluated. At bench scale, the effect of aeration and agitation on microorganism growth, and culture and induction times were evaluated. The highest enzyme activity levels at shake scale were observed in 12 h culture after 2–4 h induction. At bench scale the highest enzyme activity levels were observed after 2 h induction. rGALNS amounts in inclusion bodies fraction were up to 17-fold higher than those observed in the soluble fraction. However, the highest levels of active enzyme were found in the soluble fraction. Western blot analysis showed the presence of a 50-kDa band, in both soluble and inclusion bodies fractions. These results show for the first time the feasibility and potential of production of active rGALNS in a prokaryotic system for development of enzyme replacement therapy for MPS IVA disease.     

Lactose- and galactose-utilizing strains of poly(hydroxyalkanoic acid)-accumulating Alcaligenes eutrophus and Pseudomonas saccharophila obtained by recombinant DNA technology

Summary Transposon Tn 951-encoded -galactosidase was expressed in Pseudomonas saccharophila and enabled this bacterium to grow on lactose as sole carbon source. In contrast, -galactosidase was not expressed in Alcaligenes eutrophus even if the lacZ gene of Tn 951 was separated from the lacI gene. However, -galactosidase was expressed in A. eutrophus, if a DNA fragment, which was suspected to harbour the promoter of the A. eutrophus poly(3-hydroxybutyric acid)-synthetic genes, was ligated to the promoter probe vector pMC1403, which employs lac Z, Y as reporter genes. Plasmid pPL76, which harboured one of the promoter-lac fusions, enabled A. eutrophus not only to express -galactosidase but also to grow slowly on lactose (doubling time &#x003D; 25–30 h). Subsequently, the promoter-lac fusion was ligated to Tn5 in pSUP5011 and was inserted into the genome of A. eutrophus H16 and of the glucose-utilizing mutant H16-G⁺1 by applying the suicide plasmid technique. Two recombinant strains, H16-cPL and H16-G⁺1-cPL, which grow with a doubling time of 16–23 h on lactose, were investigated in detail. The cells only utilized the glucose residue of lactose as a carbon source for grouth and excreted galactose into the medium. Only after the Escherichia coli gal operon had been cloned in vector pVK101 and had been mobilized to H16-cPL or H16-G⁺1-cPL, was lactose completely utilized; no galactose was detected in the medium and the growth yields increased twofold. Depending on the orientation of the gal operon in pVK101, the expression of galactokinase seems to be dependent either on the promoter of aminoglycoside phosphotransferase gene (kan) or on the promoter of the tetR gene. Offprint requests to: A. Steinbüchel     

Genetic Alterations at Chromosome 6 Associated with Cervical Cancer Progression

Loss of heterozygosity (LOH) analysis on chromosome 6 was performed to define the genetic changes that occur in the development of squamous cell cervical cancer (SCC). Detailed analysis with 28 microsatellite markers revealed several loci with high frequency of deletions at the short (6p25, 6p22, 6p21.3) and long (6q14, 6q16–q21, 6q23–q24, 6q25, 6q27) arms of chromosome 6. Examination of microdissected 37 SCC and 22 cervical intraepithelial neoplasias (CIN) revealed allelic deletions in the HLA class I–III region (6p22–p21.3) and at subtelomeric locus 6p25-ter in more than 40% of CIN. By a combination of LOH and microdissection of multiple samples from the same tumor sections, we studied the intratumoral genetic heterogeneity of SCC, and identified clonal and subclonal allelic deletions. Half of SCC had clonal allelic deletion at D6S273, which is localized in intron of Ly6G6D (MEGT1) gene mapped in the HLA class III region. The LOH frequency at 6q in CIN cases did not exceed 20%. Allelic deletions at two loci, 6q14 and 6q16–q21, were for the first time associated with invasion and metastasis in SCC.     

Genome-wide linkage and peak-wide association study of obesity-related quantitative traits in Caribbean Hispanics

Although obesity is more prevalent in Hispanics than non-Hispanic whites in the United States, little is known about the genetic etiology of the related traits in this population. To identify genetic loci influencing obesity in non-Mexican Hispanics, we performed a genome-wide linkage scan in 1,390 subjects from 100 Caribbean Hispanic families on six obesity-related quantitative traits: body mass index (BMI), body weight, waist circumference, waist-to-hip ratio, abdominal and average triceps skinfold thickness after adjusting for significant demographic and lifestyle factors. We then carried out an association analysis of the linkage peaks and the FTO gene in an independent community-based Hispanic subcohort (N = 652, 64% Caribbean Hispanics) from the Northern Manhattan Study. Evidence of linkage was strongest on 1q43 with multipoint LOD score of 2.45 (p = 0.0004) for body weight. Suggestive linkage evidence of LOD > 2.0 was also identified on 1q43 for BMI (LOD = 2.03), 14q32 for abdominal skinfold thickness (LOD = 2.17), 16p12 for BMI (LOD = 2.27) and weight (LOD = 2.26), and 16q23–24 for average triceps skinfold thickness (LOD = 2.32). In the association analysis of 6,440 single nucleotide polymorphisms (SNPs) under 1-LOD unit down regions of our linkage peaks on chromosome 1q43 and 16p12 as well as in the FTO gene, we found that two SNPs (rs6665519 and rs669231) on 1q43 and one FTO SNP (rs12447427) were significantly associated with BMI or body weight after adjustment for multiple testing. Our results suggest that in addition to FTO, multiple genetic loci, particularly those on 1q43 region, may contribute to the variations in obesity-related quantitative traits in Caribbean Hispanics.     

Chromosomal localization of homeobox genes and associated markers on porcine Chromosomes 3, 5, 12, 15, 16 and 18: comparative mapping study with human and mouse

Four homeobox genes that belong to the four homeobox gene clusters known in mammals have been regionally assigned to four distinct porcine chromosomes in conserved regions between human and pig. HOXA11, HOXB6, HOXC8, and HOXD4 genes were mapped by radioactive in situ hybridization to porcine Chromosomes (Chrs) 18q21-24 (with a secondary signal in 16q14-21), 12p11-12, 5p11-12, and 15q22-23 respectively. Besides, we have also revealed the presence of a porcine homeobox (pig Hbx24) which, although showing DNA sequence homology with a mouse gene of HOXB cluster, was located on porcine Chr 3 (3p14-13) outside the Hox clusters. To support the identity of the homeobox gene clusters analyzed and in the light of the high sequence similarity among homeobox genes, we also localized markers known to be mapped near each Hox cluster in human. In this way, four genes were also mapped in pig: GAPD (5q12-21), GAD1 (15q21-22), INHBA (18q24), and IGFBP3 (18q24). Mapping of HOXA11, INHBA, and IGFBP3 on pig Chr 18 constitutes the first assignments of genes on this small chromosome. These new localizations extend the information on the conservation of four human chromosomal regions in the pig genome. Received: 7 August 1995 / Accepted: 16 October 1995     

Molecular basis for erythrocyte Le(a+b+) and salivary ABH partial-secretor phenotypes: expression of a FUT2 secretor allele with an A→T mutation at nucleotide 385 correlates with reducedα(1,2) fucosyltransferase activity

TheSe ^wA385T mutation of the FUT2 gene was found to correlate with both the erthrocyte Le(a+b+) and/or salivary ABH partial-secretor phenotypes of Polynesians. Constructs with FUT1 and FUT2 wild type genes, and the FUT2Se ^wA385T,se ^G428A andse ^C571T mutated alleles, were cloned into pcDNAI, and expressed in COS-7 cells. COS-7 cells transfected with theSe ^wA385T allele had weak, but detectable, (1,2)fucosyltransferase activity, with an acceptor substrate pattern similar to the wild type FUT2 gene. Comparative kinetic studies from cell extracts with mutatedSe ^wA385T and wild type FUT2 alleles gave similarK _m values, but less enzyme activity was present in cells transfected withSe ^wA385T (V _max 230 pmol h^–1 mg^–1), as compared to those transfected with FUT2 (V _max 1030 pmol h^–1 mg^–1), suggesting that the mutated enzyme is more unstable. These results confirm that the molecular basis for the erythrocyte Le(a+b+) and the associated ABH salivary partial-secretor phenotype, is an amino acid change of Ile 129Phe in the secretor (1,2)fucosyltransferase.Abbreviations (1,3/1,4)fucosyltransferase GDP-L-fucose:-D-N-acetylglucosaminide 3/4--L-fucosyltransferase - (1,2)fucosyltransferase GDP-L-fucose: -D-galactoside-2--L-fucosyltransferase - bp base pairs - FUT1 H gene; FUT2,Se gene - FUT3 Lewis gene or Fuc-TIll gene - FUT4 Fuc-TIV gene - FUT5 Fuc-TV gene - FUT6 Fuc-TVI gene - MAb monoclonal antibody - PCR polymerase chain reaction - RFLP restriction fragment length polymorphism - se ^G428A FUT2 nonsecretor GA mutation at nucleotide 428 - se ^C571T FUT2 nonsecretor CT mutation at nucleotide 571 - Se ^wA385T FUT2 secretor weak AT mutation at nucleotide 385 - SSP sequence specific primer     

(Z)-9-tetradecenal: a potent inhibitor of pheromone-mediated communication in the oriental tobacco budworm moth,Helicoverpa assulta

The behavioural significance of (Z)-9-tetradecenal to male H. assulta was tested by comparing the number of moths attracted to lures containing a standard synthetic female sex pheromone with lures in which (Z)-9-tetradecenal was also added. The standard pheromone mixture used contained 1000 g (Z)-9-hexadecenal, 50 g (Z)-11-hexadecenal, 300 g (Z)-9-hexadecenyl acetate and 15 g (Z)-11-hexadecenyl acetate impregnated on rubber septa. Addition of (Z)-9-tetradecenal to the standard pheromone was shown to significantly reduce the caught of male H. assulta when added in amounts greater than 10 g or 1% of the major pheromone component in both field and net-house experiments. The reduction in catch was found to be dependent on the quantity of (Z)-9-tetradecenal added to the standard pheromone. The implications of these results on conspecific and inter-specific pheromone-mediated communication in H. assulta and related sympatric heliothine species is discussed.Abbreviations Z9-16:AL (Z)-9-hexadecenal - Z11-16:AL (Z)-11-hexadecenal - Z9-16:AC (Z)-9-hexadecenyl acetate - Z11-16:AC (Z)-11-hexadecenyl acetate - Z9-14:AL (Z)-9-tetradecenal - Z9-16:OH (Z)-9-hexadecen-1-ol - Z11-16:OH (Z)-11-hexadecen-1-ol - RH relative humidity     

Stratification by CARD15 variant genotype in a genome-wide search for inflammatory bowel disease susceptibility loci

Previously we have conducted a genome-wide search for inflammatory bowel disease susceptibility loci in a large European cohort. Results from this study demonstrated suggestive evidence of linkage to loci at chromosomes 1q, 6p, and 10p and replicated linkages on chromosomes 12 and 16. Recently, NOD2/CARD15 on chromosome 16q12 has been found to be strongly associated with Crohn's disease. In order to determine if there are other loci in the genome that interact with the three associated functional variants in CARD15 (R702W, G908R, 1007fs), we have stratified our large inflammatory bowel disease genome scan cohort by dividing pedigrees into two groups stratified by CARD15 variant genotype. The two pedigree groups were analysed using non-parametric allele sharing methods. The group of pedigrees that contained one of the three CARD15 variants had two suggestive linkage results occurring in 6p (lod = 3.06 at D6S197, IBD phenotype) and 10p (lod=2.29 at D10S197, CD phenotype). In addition, at 16q12 where CARD15 is located, the original genome scan had a peak lod score of 2.18 at D16S415 (CD phenotype). The stratified pedigree cohort containing one of three CARD15 variants had a peak lod score of 0.90 at D16S415 (CD phenotype), accounting for approximately less than half of the genetic evidence for linkage at this locus. This result is in agreement with the existence of a substantial number of private variants at the NOD2/CARD15 locus. Interaction with NOD2/CARD15 needs to be considered in future gene identification efforts on chromosomes 6 and 10.     

Systemic lupus erythematosus susceptibility loci defined by genome scan meta-analysis

To date, several susceptibility loci for systemic lupus erythematosus (SLE) have been identified by individual genome-wide scans, but many of these loci have shown inconsistent results across studies. Additionally, many individual studies are at the lower limit of acceptable power recommended for declaring significant linkage. The genome search meta-analysis (GSMA) has been proposed as a valid and robust method for combining several genome scan results. The aim of this study is to investigate whether there is any consistent evidence of linkage across multiple studies, and to identify novel SLE susceptibility loci by using GSMA method. Twelve genome scan results generated from nine independent studies have been used for the present GSMA. All together, the data consists of 605 families with 1,355 SLE affected individuals from three self-reported ethnicities; Caucasian, African-American, and Hispanic. For each study, the genome was divided into 120 bins (30 cM) and ranked according to the maximum evidence of linkage within each bin. The ranks were summed and averaged across studies following which the significance was assessed by the permutation tests. The present study identified two genomic locations at 6p22.3–6p21.1 and 16p12.3–16q12.2 that met genome-wide significance (p<0.000417). The identified region at 6p22.3–6p21.1 contains the HLA region. The combined p-values using Fisher’s method also supported the significance in these regions. Clustering of significant adjacent bins was observed for chromosomes 6 and 16. Additionally, there are 12 other bins with two point-wise p-values (Psumrnk and Pord) <0.05, suggesting that these bin regions are highly likely to contain SLE susceptibility loci. Among them, present GSMA also identified two novel regions at 4q32.1–4q34.3 and 13q13.2–13q22.2. However, separate analysis using only Caucasian populations identified the strongest evidence for linkage at chromosome 6p21.1–6q15 (Psumrnk=0.00021). One interesting novel region suggests that 3q22.1–3q25.33 (Psumrnk=0.01376) may be an ethnicity-specific SLE linkage. In summary, the present GSMA have identified two statistically significant genomic regions that reconfirmed the SLE linkage at chromosomes 6 and 16.     

Cytochromes,rubredoxin, and sulfur metabolism of the non-thiosulfate-utilizing green sulfur bacterium Pelodictyon luteolum

Two soluble c-type cytochromes (c-553 and c-555) and the nonheme iron-containing protein rubredoxin of the non-thiosulfate-utilizing green sulfur bacterium Pelodictyon luteolum were highly purified by ion exchange column chromatography, gel filtration and ammonium sulfate fractionation. Both cytochrome are small and basic hemoproteins, while rubredoxin is an acidic small nonheme iron protein. Cytochrome c-553 has a molecular weight of 13,000 determined by Sephacryl S-200 chromatography and of 10,700 by electrophoresis on SDS acrylamide gel, an isoelectric point at pH 10.2, a redox-potential of +220 mV. It shows maxima at 413 nm in the oxidized form, and the characteristic three maxima in the reduced state (-band at 553 nm, -band at 523 nm, -band at 417 nm). The best purity index (A ₂₈₀/A ₄₁₇) obtained was 0.18. Cytochrome c-555 (best purity index obtained: A ₂₈₀/A ₄₁₈=0.17) has an isoelectric point at pH 10.5, a molecular weight of 9,500 (by electrophoresis on SDS acrylamide gel) and a redox-potential of +160mV. The reduced form of this cytochrome shows the typical bands of c-type cytochromes at 555 (551) nm (-band), 523 nm (-band) and 418 nm (-band), while the oxidized form has the -band at 413 nm.Rubredoxin (best purity index obtained: A ₂₈₀/A ₄₉₀=3.5) is an acidic small protein. Its molecular weight estimated by gel filtration and SDS acrylamide gel electrophoresis is 27,000 and 6,300 respectively. The monomer of this protein contains one iron atom per molecule. Rubredoxin has an isoelectric point at pH 2.8 and shows maxima at 570 nm, 490 nm and 370 nm in the oxidized form.During anaerobic sulfide oxidation of a growing culture of Pelodictyon luteolum elemental sulfur is the first main product, which appears in the medium. Elemental sulfur is further oxidized to sulfate, after the available sulfide is completely consumed by the cells.Non-common abbreviations C Chlorobium - P Pelodictyon - SDS sodium dodecylsulfate - HIPIP high-potential-iron-sulfur-protein Offprint requests to: U. Fischer