Association of HLA-DRB1 amino acid residues with giant cell arteritis: genetic association study,meta-analysis and geo-epidemiological investigation

IntroductionGiant cell arteritis (GCA) is an autoimmune disease commonest in Northern Europe and Scandinavia. Previous studies report various associations with HLA-DRB1*04 and HLA-DRB1*01; HLA-DRB1 alleles show a gradient in population prevalence within Europe. Our aims were (1) to determine which amino acid residues within HLA-DRB1 best explained HLA-DRB1 allele susceptibility and protective effects in GCA, seen in UK data combined in meta-analysis with previously published data, and (2) to determine whether the incidence of GCA in different countries is associated with the population prevalence of the HLA-DRB1 alleles that we identified in our meta-analysis.MethodsGCA patients from the UK GCA Consortium were genotyped by using single-strand oligonucleotide polymerization, allele-specific polymerase chain reaction, and direct sequencing. Meta-analysis was used to compare and combine our results with published data, and public databases were used to identify amino acid residues that may explain observed susceptibility/protective effects. Finally, we determined the relationship of HLA-DRB1*04 population carrier frequency and latitude to GCA incidence reported in different countries.ResultsIn our UK data (225 cases and 1378 controls), HLA-DRB1*04 carriage was associated with GCA susceptibility (odds ratio (OR) = 2.69, P = 1.5×10⁻¹¹), but HLA-DRB1*01 was protective (adjusted OR = 0.55, P = 0.0046). In meta-analysis combined with 14 published studies (an additional 691 cases and 4038 controls), protective effects were seen from HLA-DR2, which comprises HLA-DRB1*15 and HLA-DRB1*16 (OR = 0.65, P = 8.2×10⁻⁶) and possibly from HLA-DRB1*01 (OR = 0.73, P = 0.037). GCA incidence (n = 17 countries) was associated with population HLA-DRB1*04 allele frequency (P = 0.008; adjusted R² = 0.51 on univariable analysis, adjusted R² = 0.62 after also including latitude); latitude also made an independent contribution.ConclusionsWe confirm that HLA-DRB1*04 is a GCA susceptibility allele. The susceptibility data are best explained by amino acid risk residues V, H, and H at positions 11, 13, and 33, contrary to previous suggestions of amino acids in the second hypervariable region. Worldwide, GCA incidence was independently associated both with population frequency of HLA-DRB1*04 and with latitude itself. We conclude that variation in population HLA-DRB1*04 frequency may partly explain variations in GCA incidence and that HLA-DRB1*04 may warrant investigation as a potential prognostic or predictive biomarker.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0692-4) contains supplementary material, which is available to authorized users.     

Phylogenetic surveillance of viral genetic diversity and the evolving molecular epidemiology of human immunodeficiency virus type 1

With ongoing generation of viral genetic diversity and increasing levels of migration, the global human immunodeficiency virus type 1 (HIV-1) epidemic is becoming increasingly heterogeneous. In this study, we investigate the epidemiological characteristics of 5,675 HIV-1 pol gene sequences sampled from distinct infections in the United Kingdom. These sequences were phylogenetically analyzed in conjunction with 976 complete-genome and 3,201 pol gene reference sequences sampled globally and representing the broad range of HIV-1 genetic diversity, allowing us to estimate the probable geographic origins of the various strains present in the United Kingdom. A statistical analysis of phylogenetic clustering in this data set identified several independent transmission chains within the United Kingdom involving recently introduced strains and indicated that strains more commonly associated with infections acquired heterosexually in East Africa are spreading among men who have sex with men. Coalescent approaches were also used and indicated that the transmission chains that we identify originated in the late 1980s to early 1990s. Similar changes in the epidemiological structuring of HIV epidemics are likely to be taking in place in other industrialized nations with large immigrant populations. The framework implemented here takes advantage of the vast amount of routinely generated HIV-1 sequence data and can provide epidemiological insights not readily obtainable through standard surveillance methods.     

Prevalence of CADASIL and Fabry Disease in a Cohort of MRI Defined Younger Onset Lacunar Stroke

Background and Purpose

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), caused by mutations in the NOTCH3 gene, is the most common monogenic disorder causing lacunar stroke and cerebral small vessel disease (SVD). Fabry disease (FD) due to mutations in the GLA gene has been suggested as an underdiagnosed cause of stroke, and one feature is SVD. Previous studies reported varying prevalence of CADASIL and FD in stroke, likely due to varying subtypes studied; no studies have looked at a large cohort of younger onset SVD. We determined the prevalence in a well-defined, MRI-verified cohort of apparently sporadic patients with lacunar infarct.

Methods

Caucasian patients with lacunar infarction, aged ≤70 years (mean age 56.7 (SD8.6)), were recruited from 72 specialist stroke centres throughout the UK as part of the Young Lacunar Stroke DNA Resource. Patients with a previously confirmed monogenic cause of stroke were excluded. All MRI’s and clinical histories were reviewed centrally. Screening was performed for NOTCH3 and GLA mutations.

Results

Of 994 subjects five had pathogenic NOTCH3 mutations (R169C, R207C, R587C, C1222G and C323S) all resulting in loss or gain of a cysteine in the NOTCH3 protein. All five patients had confluent leukoaraiosis (Fazekas grade ≥2). CADASIL prevalence overall was 0.5% (95% CI 0.2%-1.1%) and among cases with confluent leukoaraiosis 1.5% (95% CI 0.6%-3.3%). No classic pathogenic FD mutations were found; one patient had a missense mutation (R118C), associated with late-onset FD.

Conclusion

CADASIL cases are rare and only detected in SVD patients with confluent leukoaraiosis. No definite FD cases were detected.     

Long-term proliferation of mouse thymic epithelial cells in culture

Summary Epithelial cells from the normal mouse thymus were successfully cultivated on tissue culture plastic when plated with lethally irradiated support cells of the LA7 rat mammary tumor line. As the irradiated LA7 cells slowly decreased in number the thymus cells proliferated concomitantly to form a confluent monolayer. The cells now in culture have been subcultured 8 times, have doubled in number at least 30 times, and are still proliferating vigorously. The culture technique also supported clonal growth from a single cell, and nine clones have been isolated. The colony-forming efficiency of thymic cells plated at low concentrations was about 8%. These cultures were never overgrown by fibroblasts. The thymus cells were characterized as epithelial by the presence of cytoplasmic keratin and numerous desmosomes and tonofilaments. They were shown to be mouse cells by immunocytochemistry with species specific antibodies, by isoenzyme analysis, and by karyology. The cells stained when reacted with antibodies to tubulin, vimentin, and actin, but not with antibodies to Thy-1.2, Lyt-1, Lyt-2, Ia, or H-2 proteins. More than 85% of the cells had a normal mouse diploid chromosome number of 40. This culture technique opens the way for future studies of T-cell education with homogeneous thymic epithelial cell populations both in vitro and after reimplantation into genetically defined strains of mice. This work was supported by the Veterans Administration, Washington, D.C.     

Proteolysis in eucaryotic cells: Aminopeptidases and dipeptidyl aminopeptidases of yeast revisited

Using nine different l-aminoacyl-4-nitroanilides and four different dipeptidyl-4-nitroanilides, aminopeptidases and dipeptidyl aminopeptidases active at pH 7.5 and (or) pH 5.5 in logarithmically growing and stationary-phase cells of Saccharomyces cerevisiae were searched for. Ion-exchange chromatography was used to separate the proteins of the soluble cell extract. Besides the three already-characterized aminopeptidases—aminopeptidase I (P. Matile, A. Wiemken, and W. Guyer (1971) Planta (Berlin)96, 43–53; J. Frey and K. H. Röhm (1978) Biochim. Biophys. Acta527, 31–41), aminopeptidase II (J. Frey and K. H. Röhm (1978) Biochim. Biophys. Acta527, 31–41; J. Knüver (1982) Thesis, Fachbereich Chemie, Marburg, FRG), and aminopeptidase Co (T. Achstetter, C. Ehmann, and D. H. Wolf (1982) Biochem. Biophys. Res. Commun.109, 341–347)—12 additional aminopeptidase activities are found in soluble cell extracts eluting from the ion-exchange column. These activities differ from the characterized aminopeptidases in one or more of the parameters such as charge, size, substrate specificity, inhibition pattern, pH optimum for activity and regulation. Also, a particulate aminopeptidase, called aminopeptidase P, is found in the nonsoluble fraction of disintegrated cells. Besides the described particulate X-prolyl-dipeptidyl aminopeptidase (M. P. Suarez Rendueles, J. Schwencke, N. Garcia-Alvarez and S. Gascon (1981) FEBS Lett.131, 296–300), three additional dipeptidyl aminopeptidase activities of different substrate specificities are found in the soluble extract.     

Reconstitution of maltose transport in Escherichia coli: conditions affecting import of maltose-binding protein into the periplasm of calcium-treated cells.

The reconstitution of active transport by the Ca2+ -induced import of exogenous binding protein was studied in detail in whole cells of a malE deletion mutant lacking the periplasmic maltose-binding protein. A linear increase in reconstitution efficiency was observed by increasing the Ca2+ - concentration in the reconstitution mixture up to 400 mM. A sharp pH optimum around pH 7.5 was measured for reconstitution. Reconstitution efficiency was highest at 0 degree C and decreased sharply with increasing temperature. The time necessary for optimal reconstitution at 0 degree C and 250 mM Ca2+ was about 1 min. The competence for reconstitution was highest in exponentially growing cultures with cell densities up to 1 X 10(9)/ml and declined when the cells entered the stationary-growth phase. The apparent Km for maltose uptake was the same as that of wild-type cells (1 to 2 microM). Vmax at saturating maltose-binding protein concentration was 125 pmol per min per 7.5 X 10(7) cells (30% of the wild-type activity). The concentration of maltose-binding protein required for half-maximal reconstitution was about 1 mM. The reconstitution procedure appears to be generally applicable. Thus, galactose transport in Escherichia coli could also be reconstituted by its respective binding protein. Maltose transport in E. coli was restored by maltose-binding protein isolated from Salmonella typhimurium. Finally, in S. typhimurium, histidine transport was reconstituted by the addition of shock fluid containing histidine-binding protein to a hisJ deletion mutant lacking histidine-binding protein. The method is fast and general enough to be used as a screening procedure to distinguish between transport mutants in which only the binding protein is affected and those in which additional transport components are affected.     

Differential usage of photoreceptors for chalcone synthase gene expression during plant development

Photoregulation of chalcone synthase (CHS) mRNA accumulation was analysed in parsley (Petroselinum crispum) and mustard (Sinapis alba) plants at different developmental stages. In both species, CHS mRNA accumulation in young etiolated seedlings was primarily under phytochrome control. In leaves of adult re-etiolated plants, a UV-B photoreceptor was predominantly involved in photocontrol. The reduced red light control in mature leaves was not due to the absence of immunoreactive phytochrome. The apparent dependence of photoreceptor usage on the developmental state of the cell or organism was in accordance with observations on the photoregulation of fusion constructs between CHS promoters from parsley or mustard and the β-glucuronidase reporter gene (GUS). When tested in the parsley protoplast transient expression system, both constructs yielded the same type of photoregulation as observed for the endogenous CHS gene.     

New proteolytic enzymes in yeast

Yeast mutants lacking three proteolytic enzymes—proteinase B, carboxypeptidase Y, and carboxypeptidase S—have been constructed. Search for new proteolytic activities in these mutants with the aid of chromogenic peptide substrates developed for serum proteinases led to the detection of new proteolytic activities, active in the neutral pH range. Sephadex chromatography of a 100,000g supernate of mutant extracts, tests against four different substrates, and partial characterization of their sensitivity to various inhibitors indicate multiple activities. Two activities, called proteinase M and proteinase P, were found in the sedimentable membranous fraction of mutant extracts.     

N-(ϱ-coumaryl)-tryptamine and N-ferulyl tryptamine in kernels of Zea mays

N-(?-coumaryl)tryptamine and N-ferulyltryptamine were isolated from aqueous acetone extracts of ground kernels of Zea mays by successive column chromatography on partially sulfonated styrenedivinylbenzene copolymer resin, lipophilic Sephadex and preparative TLC. Identification of these compounds was made by GCMS of their trimethylsilyl derivatives and the trimethylsilyl derivatives of their acid hydrolysis products.