Past environmental sun exposure and risk of multiple sclerosis: a role for the Cdx-2 Vitamin D receptor variant in this interaction.

Multiple studies have provided evidence for an association between reduced sun exposure and increased risk of multiple sclerosis (MS), an association likely to be mediated, at least in part, by the vitamin D hormonal pathway. Herein, we examine whether the vitamin D receptor (VDR), an integral component of this pathway, influences MS risk in a population-based sample where winter sun exposure in early childhood has been found to be an important determinant of MS risk. Three polymorphisms within the VDR gene were genotyped in 136 MS cases and 235 controls, and associations with MS and past sun exposure were examined by logistic regression. No significant univariate associations between the polymorphisms, rs11574010 (Cdx-2A > G), rs10735810 (Fok1T > C), or rs731236 (Taq1C > T) and MS risk were observed. However, a significant interaction was observed between winter sun exposure during childhood, genotype at rs11574010, and MS risk (P = 0.012), with the 'G' allele conferring an increased risk of MS in the low sun exposure group (

Melanocortin 1 receptor genotype, past environmental sun exposure, and risk of multiple sclerosis.

OBJECTIVE: Low past sun exposure, fair skin type, and polymorphisms of the MC1R gene have been associated with multiple sclerosis (MS) risk. We aimed to investigate the interplay between melanocortin 1 receptor gene variants, red hair/fair skin phenotype, and past environmental sun exposure in MS. METHODS: Population-based case-control study in Tasmania, Australia, involving 136 cases with MS and 272 controls randomly drawn from the community and matched on sex and year of birth. Measures included past sun exposure by calendar and questionnaire, spectrophotometric skin type, and MC1R genotype, with any MC1R Arg151Cys, Arg160Trp, or Asp294His alleles present denoted as red hair color (RHC) variant. RESULTS: The association between RHC variant genotype and MS was more evident for women (odds ratio 2.02 [1.15-3.54]) than for men (odds ratio 0.65 [0.27-1.57]) (difference in effect, p = 0.03). The RHC variant genotype was associated with behavioral sun avoidance. In addition, increasing summer sun exposure at ages 6 through 10 years was associated with reduced MS risk among those with no RHC variant (p = 0.03), but not among those with RHC variant genotype (p = 0.15; difference in effect, p = 0.02). Similar findings were evident for other past sun exposure measures and when the sample was restricted to women only. CONCLUSION: The interplay between red hair color variant genotype, red hair/fair skin phenotype, and multiple sclerosis (MS) is complex. The modification of past sun exposure by MC1R genotype provides further support that ultraviolet radiation or derivatives such as vitamin D may be causally related to a reduced MS risk.

Association between birth weight and adolescent systolic blood pressure in a caucasian birth cohort differs according to skin type, CRH promoter or 11beta-HSD2 genotype.

OBJECTIVE: To examine whether the inverse association between birth weight and blood pressure varies by skin pigmentation and/or related genotypes. STUDY DESIGN: 671 children from a predominantly caucasian birth cohort were followed-up to adolescence (mean (SD) age 14.4 (0.64)). METHODS: Data on birth weight, socioeconomic status, maternal antenatal smoking, adolescent blood pressure and polymorphisms of candidate genes were obtained and analysed by multiple linear regression. RESULTS: An increase in birth weight of 1 kg was associated with an non-significant difference in adolescent systolic blood pressure of -0.53 mm Hg (95% CI -1.72 to 0.66) per kg after adjustment for child age and cohort entry criteria. The inverse association between birth weight and systolic blood pressure was stronger for those with darker skin (> or =2% melanin) (difference in effect, p = 0.02), those with more copies of the C allele of corticotropin-releasing hormone (CRH) +T1273C (p = 0.06), and those with more copies of the short (< or =236 bp) form of the 11beta-HSD2{CA}n(repeat) microsatellite (p = 0.03). CONCLUSIONS: These findings add to the evidence that cortisol-related pathways may account for at least part of the observed birth weight-blood pressure associations.

Investigation of crystallin genes in familial cataract, and report of two disease associated mutations.

AIMS: Mutations of seven crystallin genes have been shown to cause familial cataract. The authors aimed to identify disease causing crystallin mutations in paediatric cataract families from south eastern Australia. METHODS: 38 families with autosomal dominant or recessive paediatric cataract were examined. Three large families were studied by linkage analysis. Candidate genes at regions providing significant LOD scores were sequenced. Single stranded conformational polymorphism (SSCP) analysis was used to screen five crystallin genes in the probands, followed by direct sequencing of observed electrophoretic shifts. Mutations predicted to affect the coding sequence were subsequently investigated in the entire pedigree. RESULTS: A LOD score of 3.72 was obtained at the gamma-crystallin locus in one pedigree. Sequencing revealed a P23T mutation of CRYGD, found to segregate with disease. A splice site mutation at the first base of intron 3 of the CRYBA1/A3 gene segregating with disease was identified by SSCP in another large family. Five polymorphisms were also detected. CONCLUSIONS: Although mutations in the five crystallin genes comprehensively screened in this study account for 38% of paediatric cataract mutations in the literature, only two causative mutations were detected in 38 pedigrees, suggesting that crystallin mutations are a relatively rare cause of the cataract phenotype in this population.

Investigation of chromosome 2q in osteoarthritis of the hand: no significant linkage in a Tasmanian population.

BACKGROUND: Previous studies have suggested a strong genetic component to osteoarthritis (OA), especially that of the hand, and three linkage studies have suggested the existence of susceptibility loci in disparate regions of chromosome 2q. OBJECTIVE: To examine for linkage to 2q in a Tasmanian population of women and men with familial hand OA. METHODS: Hand OA (distal interphalangeal, carpometacarpal, and Heberden's nodes) was assessed by a combination of hand photographs and radiographs. A non-parametric linkage (NPL) analysis was performed on chromosome 2q of 69 members in 22 families with severe distal interphalangeal joint OA using Genehunter. A quantitative trait linkage analysis of a larger group of 456 members in 68 families was also performed using SOLAR. RESULTS: The maximum non-parametric linkage score was 1.05 (p=0.15) at marker IL1R1, close to the centromere. All components of hand OA scores had significant heritability in this dataset (28%-35%, all p<0.001). Despite this, the quantitative trait analysis (after adjustment for age and, where appropriate, sex) yielded maximum LOD scores of 0.90 for Heberden's nodes (both sexes combined), and 1.19 for carpometacarpal OA score (women only). CONCLUSIONS: These results do not provide confirmation of linkage on chromosome 2q in the larger white population with hand OA. They suggest that there are regional variations in the genetic cause of hand OA and that other loci not on 2q may be important in this disease.

Retinopathy of prematurity: recent advances in our understanding.

Retinopathy of prematurity (ROP) has been recognised as an important cause of childhood visual impairment and blindness since the 1940s when improved facilities and treatment increased the survival rate of premature infants. Although its incidence and severity have been decreasing in developed countries over the past two decades, both are increasing in developing nations. ROP is consequently targeted as an important but avoidable disease. This review provides an updated summary and discussion of much of the work that has been produced through population, animal, cell culture, and genetic research. The authors examine the prevalence, risk factors, and possible causes of the disease with a particular focus on genetic studies. They conclude that while significant reductions in the disease have occurred in developed countries, further research is required to fully understand and prevent the disease. In the meantime, development and implementation of appropriate screening and treatment strategies will be critical in reducing blindness in developing countries.

Retinopathy of prematurity: recent advances in our understanding.

Retinopathy of prematurity (ROP) has been recognised as an important cause of childhood visual impairment and blindness since the 1940s when improved facilities and treatment increased the survival rate of premature infants. Although its incidence and severity have been decreasing in developed countries over the past two decades, both are increasing in developing nations. ROP is consequently targeted as an important but avoidable disease. This review provides an updated summary and discussion of much of the work that has been produced through population, animal, cell culture, and genetic research. The authors examine the prevalence, risk factors, and possible causes of the disease with a particular focus on genetic studies. They conclude that while significant reductions in the disease have occurred in developed countries, further research is required to fully understand and prevent the disease. In the meantime, development and implementation of appropriate screening and treatment strategies will be critical in reducing blindness in developing countries.

Evidence for genetic heterogeneity within eight glaucoma families, with the GLC1A Gln368STOP mutation being an important phenotypic modifier.

OBJECTIVE: To investigate the phenotype and age-related penetrance of primary open-angle glaucoma (POAG) in Australian families with the most common Myocilin mutation (Gln368STOP). DESIGN: Cross-sectional genetic study. PARTICIPANTS: Eight pedigrees carrying the Gln368STOP mutation were ascertained from 1730 consecutive cases of POAG in the Glaucoma Inheritance Study in Tasmania. METHODS: Index cases and available family members were examined for signs of glaucoma, and the presence of the GLC1A Gln368STOP mutation was ascertained by single-strand conformation polymorphism analysis and subsequent direct sequencing. RESULTS: From the eight pedigrees, 29 Gln368STOP mutation-carrying individuals with either ocular hypertension (OHT) or POAG were found, with a mean age at diagnosis of 52.4 +/- 12.9 years and a mean peak intraocular pressure (IOP) of 28.4 +/- 4.7 mmHg. A further 11 mutation carriers older than 40 years have been studied, who as yet show no signs of OHT or POAG. Within the 8 pedigrees, a further 31 individuals with OHT or POAG were identified who did not carry the Gln368STOP mutation. For these individuals the mean age at diagnosis was higher (62.3 +/- 13.7 years, P < 0.01), and the mean peak IOP was lower (25.4 +/- 6.4 mmHg, P = 0.01). For Gln368STOP carriers, age-related penetrance for OHT or POAG was 72% at age 40 years and 82% at age 65 years. A positive family history of POAG was present in all index cases. Five of the eight pedigrees had a positive family history on both maternal and paternal sides. Seven of the eight pedigrees had one or more individuals with POAG who did not carry the mutation. Eight of the 29 Gln368STOP carriers with OHT or POAG had undergone trabeculectomy. CONCLUSIONS: The GLC1A Gln368STOP mutation is associated with POAG, which in the pedigrees studied is of a younger age of onset and higher peak IOP than non-mutation glaucoma cases. In addition, Gln368STOP mutation glaucoma cases were more likely to have undergone glaucoma drainage surgery. We have not observed simple autosomal dominant inheritance patterns for POAG in these pedigrees. Other factors, as yet uncharacterized, are involved in expression of the POAG phenotype in Gln368STOP pedigrees.

Laboratory methods in ophthalmic genetics: obtaining DNA from patients.

DNA samples are the fundamental research substrate in genetics. Although methodology and cost-effectiveness in molecular biology have improved dramatically, collecting biological samples and extracting DNA continue to be expensive and time-consuming steps of genetic research. This article reviews the issues surrounding the choice of biological samples for methods of DNA extraction as well as the storage and transport of biological and DNA samples for genetic studies.

Testisin, a new human serine proteinase expressed by premeiotic testicular germ cells and lost in testicular germ cell tumors.

We have cloned and characterized a cDNA encoding a new human serine proteinase, testisin, that is abundantly expressed only in the testis and is lost in testicular tumors. The testisin cDNA was identified by homology cloning using degenerate primers directed at conserved sequence motifs within the catalytic regions of serine proteinases. It is 1073 nucleotides long, including 942 nucleotides of open reading frame and a 113-nucleotide 3' untranslated sequence. Northern and dot blot analyses of RNA from a range of normal human tissues revealed a 1.4-kb mRNA species that was present only in testis, which was not detected in eight of eight testicular tumors. Testisin cDNA is predicted to encode a protein of 314 amino acids, which consists of a 19-amino acid (aa) signal peptide, a 22-aa proregion, and a 273-aa catalytic domain, including a unique 17-aa COOH-terminal hydrophobic extension that is predicted to function as a membrane anchor. The deduced amino acid sequence of testisin shows 44% identity to prostasin and contains features that are typical of serine proteinases with trypsin-like substrate specificity. Antipeptide antibodies directed against the testisin polypeptide detected an immunoreactive testisin protein of Mr 35,000-39,000 in cell lysates from COS-7 cells that were transiently transfected with testisin cDNA. Immunostaining of normal testicular tissue showed that testisin was expressed in the cytoplasm and on the plasma membrane of premeiotic germ cells. No staining was detected in eight of eight germ cell-derived testicular tumors. In addition, the testisin gene was localized by fluorescence in situ hybridization to the short arm of human chromosome 16 (16p13.3), a region that has been associated with allellic imbalance and loss of heterozygosity in sporadic testicular tumors. These findings demonstrate a new cell surface serine proteinase, loss of which may have a direct or indirect role in the progression of testicular tumors of germ cell origin.

DNase I hypersensitive sites in the 5' flanking region of the human plasminogen activator inhibitor type 2 (PAI-2) gene are associated with basal and tumor necrosis factor-alpha-induced transcription in monocytes.

The plasminogen activator inhibitor type 2 (PAI-2) gene encodes a serine proteinase inhibitor (serpin) which is rapidly induced in response to the inflammatory cytokine, tumour necrosis factor-alpha (TNFalpha) in monocytes and macrophages. As an initial step towards understanding the molecular mechanisms underlying PAI-2 gene regulation in monocytes, we report here the analysis of the chromatin structure of 9.6 kb of 5' flanking region of the human PAI-2 gene for potential cis-acting regulatory regions using DNase I hypersensitivity mapping. Sites sensitive to DNase I were mapped in two monocytic cell lines representative of early monocytic differentiation; U937 cells, which synthesise low constitutive levels of PAI-2 that were induced following treatment with TNFalpha, and a MonoMac6 cell line which did not synthesise PAI-2 even after treatment with TNFalpha. Six DNase I hypersensitive sites (DHS) were identified; three upstream of the transcription initiation site (DH1, DH2, DH3) and three downstream of the transcription initiation site which were contained within intron A (DH4, DH5) and the exon 2/intron B junction (DH6). Among these, one distally located DH site (DH2) disappeared in both cell lines following treatment with TNFalpha. Two DH sites (DH1, DH6) were absent in PAI-2-producing U937 cells, but were present in MonoMac6 cells, which did not produce PAI-2, indicating the possible involvement of negative regulatory elements in the suppression of PAI-2 gene expression. The results demonstrate the involvement of chromatin structure in transcriptional responsiveness of the PAI-2 gene promoter and identify several loci which may be key control regions for PAI-2 gene transcription.

The serine proteinase inhibitor (serpin) plasminogen activation inhibitor type 2 protects against viral cytopathic effects by constitutive interferon alpha/beta priming.

The serine proteinase inhibitor (serpin) plasminogen activator inhibitor type 2 (PAI-2) is well characterized as an inhibitor of extracellular urokinase-type plasminogen activator. Here we show that intracellular, but not extracellular, PAI-2 protected cells from the rapid cytopathic effects of alphavirus infection. This protection did not appear to be related to an effect on apoptosis but was associated with a PAI-2-mediated induction of constitutive low-level interferon (IFN)-alpha/beta production and IFN-stimulated gene factor 3 (ISGF3) activation, which primed the cells for rapid induction of antiviral genes. This primed phenotype was associated with a rapid development of resistance to infection by the PAI-2 transfected cells and the establishment of a persistent productive infection. PAI-2 was also induced in macrophages in response to viral RNA suggesting that PAI-2 is a virus response gene. These observations, together with the recently demonstrated PAI-2-mediated inhibition of tumor necrosis factor-alpha induced apoptosis, (a) illustrate that PAI-2 has an additional and distinct function as an intracellular regulator of signal transduction pathway(s) and (b) demonstrate a novel activity for a eukaryotic serpin.

The C-D interhelical domain of the serpin plasminogen activator inhibitor-type 2 is required for protection from TNF-alpha induced apoptosis.

The serine proteinase inhibitor (serpin), plasminogen activator inhibitor type 2 (PAI-2), has been reported to inhibit tumor necrosis factor-alpha (TNF) induced apoptosis. In order to begin to understand the molecular basis for this protection, we have investigated the importance of a structural domain within the PAI-2 molecule, the C-D interhelical region, in mediating the protective effect. The C-D interhelical region is a 33 amino acid insertion which is unique among serpins and has been implicated in transglutaminase catalyzed cross-linking of PAI-2 to cell membranes. We have constructed a mutant of PAI-2 wherein 23 amino acids are deleted from the C-D interhelical region generating a structure predicted to be homologous to the closely related, but non-inhibitory serpin, chicken ovalbumin. The PAI-2Delta65/87 deletion mutant retained inhibitory activity against its known serine proteinase target, urokinase-type plasminogen activator (uPA); however expression of this mutant in HeLa cells failed to protect from TNF-induced apoptosis. Analyses of the cellular distribution of PAI-2 showed that intracellular PAI-2, and not secreted or cell-surface PAI-2, was likely responsible for the observed protection from TNF-induced apoptosis. No evidence was found for specific cross-linking of PAI-2 to the plasma membrane in either control or TNF/cycloheximide treated cells. The data demonstrate that the PAI-2 C-D interhelical domain is functionally important in PAI-2 protection from TNF induced apoptosis and suggest a novel function for the C-D interhelical domain in the protective mechanism.

Accumulation of p16CDKN2A in response to ultraviolet irradiation correlates with late S-G(2)-phase cell cycle delay.

p16 is the product of the CDKN2A locus, which is mutated or deleted in many human tumors. In response to nonlethal UVC irradiation, HeLa cells accumulate elevated levels of p16. The accumulation of p16 is delayed 8-12 h following irradiation and correlates with S-phase and G2 delays, decreasing as the cells recover and recommence normal cell growth. The maximum levels of p16 correlated with G2 delay. The UVC-induced cell cycle delay was absent in cell lines derived from HeLa that did not express p16 and in a melanoma line deleted for p16.

Plasminogen activator inhibitor type 2 inhibits tumor necrosis factor alpha-induced apoptosis. Evidence for an alternate biological function.

Plasminogen activator inhibitor type 2 (PAI-2) is a serine proteinase inhibitor or serpin that is a major product of macrophages in response to endotoxin and inflammatory cytokines. We have explored the role of PAI-2 in apoptotic cell death initiated by tumor necrosis factor alpha (TNF). HeLa cells stably transfected with PAI-2 cDNA were protected from TNF-induced apoptosis, whereas cells transfected with antisense PAI-2 cDNA, a control gene, or the plasmid vector alone remained susceptible. The level of PAI-2 expressed by different HeLa cell clones was inversely correlated with their sensitivity to TNF. Loss of TNF sensitivity was not a result of loss of TNF receptor binding. In contrast, PAI-2 expression did not confer protection against apoptosis induced by ultraviolet or ionizing radiation. The serine proteinase urokinase-type plasminogen activator was not demonstrated to be the target of PAI-2 action. The P1-Arg amino acid residue of PAI-2 was determined to be required for protection, because cells expressing PAI-2 with an Ala in this position were not protected from TNF-mediated cell death. The results suggest that intracellular PAI-2 might be an important factor in regulating cell death in TNF-mediated inflammatory processes through inhibition of a proteinase involved in TNF-induced apoptosis.

Tissue factor and plasminogen activator inhibitor expression in the differentiation of myeloid leukemic cells.

Disturbances in the regulation of the balance between the fibrinolytic and procoagulant properties of leukemic cells may contribute to the coagulopathy of acute leukemia. The coagulant response to a number of stimuli is regulated by the expression of tissue factor, but the role of the plasminogen activator inhibitors, PAI-1 and PAI-2, in contributing to the net coagulant response is not known. In this study, we have examined the production of these proteins by cultured myeloid leukemic cells arrested at different stages of differentiation. Northern blot analysis showed time-dependent and differential production of mRNA for PAI-2 and tissue factor, and to a much lesser extent, PAI-1, in response to the differentiating agent, 12-phorbol-13-myristate acetate. The capacity to synthesize PAI-2 appeared to be related to the stage of myeloid cell differentiation. Examination of the gene products by immunoblot analysis demonstrated multiple forms of PAI-2 in all myeloid cells examined. In addition, a common characteristic of all the myeloid cells was the production of a high molecular weight species of tissue factor which may be a secreted form unique to leukemic cells. Taken together, the findings demonstrate that myeloid leukemic cells are capable of generating a multicomponent coagulant response.

Sodium butyrate differentially modulates plasminogen activator inhibitor type-1, urokinase plasminogen activator, and its receptor in a human colon carcinoma cell.

Human colonic epithelium is exposed to varying levels of sodium butyrate, which is derived from the bacterial fermentation of dietary carbohydrate. Sodium butyrate has several effects on colonic tumor cells in vitro, including arrest of cell growth and differentiation. In the present study we have found that, in addition to a reduction in cellular proliferation, sodium butyrate induces the transient expression of plasminogen activator inhibitor type-1 (PAI-1) in the LIM 2405 human colonic tumor cell. Approximately 40% of the PAI-1 secreted is biologically active as judged by the formation of higher molecular weight, SDS-resistant complexes with urokinase plasminogen activator (uPA). The enhanced PAI-1 biosynthesis was accompanied by an increase in PAI-1 mRNA levels. During the same time period, the amount of secreted uPA remained relatively constant, but the level of cell associated uPA decreased slowly and was accompanied by a decrease in uPA mRNA levels. The uPA receptor is synthesized constitutively by these cells, and was down-regulated at both the protein and mRNA levels in response to sodium butyrate. The results demonstrate that sodium butyrate can alter the balance of components of the plasminogen activator system in a manner which favours net decreased plasminogen activator activity and suggests a role for sodium butyrate in the regulation of extracellular proteolysis.

Control of plasminogen-activator inhibitor type 2 gene expression in the differentiation of monocytic cells.

Plasminogen-activator inhibitor type 2 (PAI-2) is a potent and primary inhibitor of urokinase-type plasminogen activator. Its production in monocytic cells is thought to play an important role in the control of localized proteolysis at sites of invasion as occurs in the control of inflammatory processes, tumor invasion and cellular differentiation. Therefore, we have investigated the mechanisms responsible for the regulation of PAI-2 gene expression in differentiating monocytic cells using the human promyelocytic cell line, HL-60, as a model. These cells are induced to differentiate to a macrophage-like phenotype in response to phorbol ester [4-phorbol-12-myristate 13-acetate (PMA)]. The levels of PAI-2 mRNA are barely detectable in undifferentiated cells, however, activation with PMA is associated with a rapid induction of PAI-2 transcripts, reaching a maximum of 25-fold in 4 h. Nuclear run on assays demonstrate that this induction is related primarily to an enhanced rate of gene transcription. Inhibition of de novo protein synthesis by cycloheximide increases PAI-2 mRNA levels in both resting (sevenfold) and PMA-treated cells (fivefold) after 4 h, but has no detectable effect on the rate of PAI-2 gene transcription. The initial apparent half-life of the induced PAI-2 mRNA, determined by actinomycin-D-decay experiments, is very short, 32 min, suggesting rapid turnover. Furthermore, the PAI-2 mRNA transcript is stabilized in the presence of cycloheximide, with a fourfold increase in the observed half-life. The results demonstrate that PAI-2 gene expression is regulated through post-transcriptional mechanisms in undifferentiated cells, while both transcriptional and post-transcriptional events govern the level of PAI-2 transcripts in cells differentiated along the monocytic pathway. Destabilization of the PAI-2 transcript may be associated with (A + U)-rich sequences found in the 3'-untranslated region of PAI-2 mRNA. The short half life and rapid, strong induction of PAI-2 point to an important, perhaps crucial, role in the differentiation of monocyte cells.