Lack of replication of association findings in complex disease: an analysis of 15 polymorphisms in prior candidate genes for sporadic Alzheimer's disease.

There is considerable enthusiasm for the prospect of using common polymorphisms (primarily single nucleotide polymorphisms; SNPs) in candidate genes to unravel the genetics of complex disease. This approach has generated a number of findings of loci which are significantly associated with sporadic Alzheimer's disease (AD). In the present study, a total of 15 genes of interest were chosen from among the previously published reports of significant association in AD. Genotyping was performed on polymorphisms within those genes (14 SNPs and one deletion) using Dynamic Allele Specific Hybridization (DASH) in 204 Swedish patients with sporadic late-onset AD and 186 Swedish control subjects. The genes chosen for analysis were; low-density lipoprotein receptor-related protein (LRP1), angiotensin converting enzyme (DCP1), alpha-2-macroglobulin (A2M), bleomycin hydrolase (BLMH), dihydrolipoyl S-succinyltransferase (DLST), tumour necrosis factor receptor superfamily member 6 (TNFRSF6), nitric oxide synthase (NOS3), presenilin 1 (PSEN1), presenilin 2 (PSEN2), butyrylcholinesterase (BCHE), Fe65 (APBB1), oestrogen receptor alpha (ESR1), cathepsin D (CTSD), methylenetetrahydrofolate reductase (MTHFR), and interleukin 1A (IL1A). We found no strong evidence of association for any of these loci with AD in this population. While the possibility exists that the genes analysed are involved in AD (ie they have weak effects and/or are population specific), results reinforce the need for extensive replication studies if we are to be successful in defining true risk factors in complex diseases.

TNF gene polymorphism and its relation to intracerebral production of TNFalpha and TNFbeta in AD.

OBJECTIVE: To analyze the extent of tumor necrosis factor-alpha (TNFalpha) and TNFbeta gene polymorphism in patients with AD and to relate it to intrathecal levels of these cytokines. METHODS: Analyses of TNFalpha and TNFbeta gene polymorphism were performed using PCR in 52 patients with AD and in 25 control subjects, and the levels of corresponding cytokines were analyzed using ELISA. RESULTS: Patients with AD displayed significantly higher intrathecal levels of TNFalpha, but not TNFbeta, compared with the control subjects. The levels of these cytokines did not differ significantly in patients displaying different alleles of the TNF gene. CONCLUSIONS: Results indicate that increased intrathecal production of TNFalpha in AD is preferentially controlled by environmental stimuli rather than genetic makeup.

Specific expression of Epstein-Barr virus in cutaneous squamous cell carcinomas from heart transplant recipients.

BACKGROUND: We investigated a Swedish group of 114 immunosuppressed cardiac allograft patients for the occurrence of posttransplant cutaneous squamous cell carcinomas. A total of 15 tumors were detected in specimens from 5 patients. METHODS: The tumors were analyzed for the presence of Epstein-Barr virus (EBV) genomes as well as EBV-specific gene expression by using three different techniques; the polymerase chain reaction (PCR), in situ hybridization, and immunohistochemistry. The material was also tested by PCR for high-risk human papilloma virus genome. RESULTS: EBV DNA could be detected by PCR in 10 of the investigated tumors, 7 of which also expressed EBV latent membrane protein 1 and/or EBV-encoded RNAs. No EBV genomes or EBV gene products could be detected in normal skin/resection margins, available from three of the tumors investigated. All tumors were negative for high-risk human papilloma virus DNA analyzed by PCR. CONCLUSIONS: In this study, we have found a high incidence of EBV-specific expression in posttransplant cutaneous squamous cell carcinomas. These results suggest that at least some of the skin cancers developing in immunocompromised heart transplant recipients are associated with EBV.

Relation between polymerase chain reaction findings and morphological changes during cytomegalovirus infection in transplanted lung.

Cytomegalovirus (CMV) can be present as a latent or productive infection resulting in disease. The polymerase chain reaction (PCR) is a sensitive technique to document the presence of CMV (DNA). Negative reactions are indicative of its absence. The presence of CMV (DNA) was assessed longitudinally in 261 transbronchial lung biopsy (TBB) specimens from 37 patients over a 6-month period. The TBB specimens from six serologically CMV-negative recipients who received lungs from serologically CMV-negative donors never showed a positive CMV-PCR(DNA) reaction during the study. Based on a study of their TBB specimens, 10 serologically CMV-positive recipients who received lungs from serologically CMV-negative donors all developed a CMV-PCR(DNA)-positive reaction and five (50%) morphologically manifested CMV disease. The remaining 21 serologically CMV-positive recipients who received lungs from serologically CMV-positive donors all developed a CMV-PCR(DNA)-positive reaction and 15 (71%) developed CMV pneumonitis. The data show that development of a positive CMV-PCR(DNA) reaction in a TBB sample within the first month after transplantation indicates a greatly increased risk of developing CMV disease. In addition, a positive CMV-PCR(DNA) reaction preceded morphologically manifest disease on average by 2 weeks. Comparisons between TBB and bronchoalveolar lavage show the former to provide a more dependable template.

Prevalence of Epstein-Barr virus and human papillomavirus in cervical samples from women attending an STD-clinic.

A group of 91 women attending the STD-clinic, Department of Dermatovenereology, Sahlgrenska Hospital, Gothenburg, were screened for EBV DNA and HPV DNA of the cervix with the PCR-technique. Presence of EBV DNA was demonstrated in 35 (38%) women and HPV DNA in 30 (33%) women. Fourteen (15%) women had both EBV DNA and HPV DNA present. Without the colposcope 20 of these women had macroscopic signs of HPV infection on the vulva and/or vagina and 71 had no signs of infection. Presence of EBV DNA was not correlated to clinical signs of HPV infection.

Demonstration of Epstein-Barr virus DNA in acetowhite lesions of the vulva.

Acetowhite lesions in the vulva disclosing koilocytosis have been related to infection by human papilloma virus (HPV). Because of the clinical resemblance of these lesions to oral hairy leukoplakia (OHL), and EBV-manifestation, 23 women with acetowhite koilocytotic lesions in the vulva were examined. The PCR-technique was used to detect EBV DNA as well as HPV DNA in 17% of 23 patients examined. In a control group of 19 patients EBV DNA was detected in 11% and HPV DNA in 42% of cell samples from normal vulvar mucosa. EBV DNA has not previously been demonstrated in the vulvar mucosa, and this virus might be related to subclinical acetowhite lesions.

Are acetowhite lesions of the cervix correlated to the presence of Epstein-Barr virus DNA?

The purpose of this study was to investigate whether Epstein-Barr virus (EBV) is associated with acetowhite lesions of the portio cervix, demonstrating koilocytosis and/or cervical intraepithelial neoplasia (CIN) I-III. The study group comprised 37 women admitted to the Department of Gynaecology and Obstetrics, Sahlgrenska University Hospital, Göteborg because of pathological colposcopy or cytology of the portio cervix. Colposcopically, all exhibited acetowhite lesions of the portio cervix. Cells were sampled with a cytobrush for examination for EBV and human papillomavirus (HPV) DNA by polymerase chain reaction (PCR) and a biopsy was taken for histopathology. Biopsies from 5 patients positive for EBV by PCR in cervical cell samples were examined by an in situ hybridization technique for EBER (Epstein-Barr virus encoded RNA), RNAs expressed in latent EBV infection. The control group consisted of women attending the Department of Dermato-Venereology at the same hospital for STD check-up. These had a normal cytology and no signs of acetowhiteness of the portio cervix. In the study group, EBV DNA was found in 30% and HPV DNA in 51%. In the control group 57% exhibited EBV DNA and 23% HPV DNA. EBV was not found to be a predictive factor in the development of koilocytosis and/or CIN I-III. HPV was a predictive factor in acetowhite, koilocytotic lesions. No expression of EBER was found in the 5 biopsies examined by in situ hybridization.

Demonstration of Epstein-Barr virus DNA and human papillomavirus DNA in acetowhite lesions of the penile skin and the oral mucosa.

Oral hairy leukoplakia (OHL), thought to be caused by Epstein-Barr virus (EBV), shows similar histological and clinical features to human papillomavirus (HPV)-related acetowhite lesions of the vulva. We thus aimed to investigate the role of both HPV and EBV in men with acetowhite lesions of the penis. HPV but not EBV was significantly associated with penile acetowhite lesions showing koilocytosis compared with normal penile skin (12/20 versus 5/20, P < 0.02). HPV (5/20) and EBV (6/20) was detected in oral mucosa of some of these individuals. These results confirm an aetiological association between HPV and acetowhite penile lesions showing koilocytosis. HPV and EBV carriage in the oral mucosa is relatively common in young sexually active men.

[Gene amplification in viral CNS infections. Rapid diagnostic identification of herpesviruses]. / Genamplifiering vid virala CNS-infektioner. Snabb diagnostik av herpesgruppens virus.

DNA amplification with the polymerase chain reaction (PCR) technique was used as a diagnostic test on cerebrospinal fluid samples in cases where herpesvirus infection of the central nervous system (CNS) was suspected. During the period, 1992-93, 47 (8.9%) of 528 patients tested were positive for one or another of the following herpesviruses: herpes simplex virus type 1 (n = 16) or type 2 (n = 9), cytomegalovirus (n = 16), varicella-zoster virus (n = 4), or Epstein-Barr virus (n = 2). The study showed PCR to be a rapid and useful diagnostic method in clinical routine, enabling early antiviral intervention in several cases with an atypical clinical picture. Moreover, cytomegalovirus was found to be an important CNS pathogen in addition to herpes simplex virus, especially during childhood.

Expression of a second Epstein-Barr virus-determined nuclear antigen in mouse cells after gene transfer with a cloned fragment of the viral genome.

Large Epstein-Barr virus (EBV) DNA restriction fragments corresponding to regions transcribed in transformed, proliferating cells were cloned in a cosmid derivative of the dominant-acting selection vector pSV2-gpt. Recombinant vectors carrying the EcoRI A fragment of EBV DNA were modified in the region corresponding to the deletion of the virion DNA in the non-transforming viral substrain P3HR-1, to create a series of recombinants lacking parts of this region. The recombinant vectors were introduced into 3T3 mouse fibroblasts under selective conditions, and resistant clones shown to contain EBV DNA sequences were analyzed for the expression of EBV-related antigens detectable by direct, indirect, and anticomplement immunofluorescence techniques. Cells that contained the BamHI K fragment expressed the EBV-determined nuclear antigen (EBNA) as expected. Cells transfected with recombinant vectors containing the BamHI W, Y, and H fragment part of the EcoRI A fragment also express a nuclear antigen detectable with certain anti-EBNA-positive human sera in anticomplement immunofluorescence tests. The BamHI WYH-induced EBNA polypeptide is similar in size to the EBNA2 polypeptide in Raji cells, as shown by gel electrophoresis and immunoblotting. The antigen is not detected in cells transfected with EcoRI A-derived vectors in which the BamHI H fragment has been deleted or in cells transformed with vectors carrying the BamHI H fragment alone. Direct and indirect immunofluorescence did not reveal the presence of antigens associated with productive infection in any of the EBV DNA-transfected fibroblast clones.

Identification of sequences in Epstein-Barr virus DNA required for the expression of the second Epstein-Barr virus-determined nuclear antigen in COS-1 cells.

The BamHI WYH region of the Epstein-Barr virus (EBV) genome encodes a protein localized to the nucleus of the infected cell, the EBV-determined nuclear antigen EBNA2. We have constructed a series of recombinant vectors that carried the complete EBNA2 gene, or the gene modified so as to contain defined deletions involving presumed exons and regulatory elements of the gene. The recombinant vectors were transfected into COS-1 cells which permit the replication of simian virus 40 origin-containing plasmids to a high copy number, and the transient expression of EBNA2 was analysed. A recombinant plasmid that carried a BglII-NotI subfragment of the BamHI WYH region (nucleotides 44664 to 50628) contained all the information necessary for inducing the expression of a full length EBNA2 polypeptide. Moreover, EBV DNA sequences between nucleotides 45,442 and 48,337 could be deleted without interfering with the ability of the vectors to induce EBNA2. On the other hand the loss of the left one-third of the BglII-NotI fragment completely abolished the EBNA2-inducing capacity of the vector. A rightward promoter consensus sequence in the BamHI W part of the BglII-NotI fragment was functional in COS-1 cells expressing EBNA2 and essential for EBV-specific RNA synthesis. The results indicated that transcription of the EBNA2 gene was initiated in the BamHI W fragment, that the transcript was spliced and that all of EBNA2 was encoded within the continuous long open reading frame in the BamHI Y and H fragments.

Detection of cytomegalovirus DNA in cerebrospinal fluid in immunocompetent patients as a sign of active infection.

Detection of cytomegalovirus (CMV) DNA by the polymerase chain reaction (PCR) in samples of cerebrospinal fluid (CSF) has been shown to be a sensitive method of diagnosing CMV disease in the central nervous system. Since CMV causes latent infection in white blood cells, an unanswered question is whether detection of latent CMV DNA in the cell fraction of CSF samples by PCR is possible in seropositive patients. In a prospective study, the finding of CMV DNA in CSF of CMV seropositive patients with suspected viral infection of the central nervous system (CNS) was evaluated clinically. Fractionation of 64 CSF samples from seropositive patients was carried out before analysing the samples for CMV DNA by PCR. In four of the five patients who had CMV DNA in the cell pellet and/or supernatant, the clinical data suggested CMV-associated neurological disease. The remaining 59 samples were negative in both pellet and supernatant. In addition, 11 CSF samples with high cell counts from patients with bacterial meningitis were examined for CMV DNA and found to be negative in 10 patients and positive in 1. One hundred thirty two uncentrifuged CSF samples were used as negative controls. The results of the study indicate that detection of CMV DNA in CSF samples by PCR correlated well with disease and was not due to latent CMV infection.

The 5' flanking region of the gene for the Epstein-Barr virus-encoded nuclear antigen 2 contains a cell type specific cis-acting regulatory element that activates transcription in transfected B-cells.

We have recently identified the promoter that positions the initiation (cap) site for RNA encoding the Epstein-Barr virus (EBV) determined nuclear antigen 2 (EBNA2) in transfected COS-1 cells. The cells were transfected with recombinant vectors that contained the BamHI WYH region of the EBV genome. In order to delineate regulatory DNA sequences required for the expression of EBNA2 the 5' flanking region of the gene was linked to reporter genes in expression vectors and transfected into EBV genome-negative lymphoid DG75 cells. We demonstrate that several cis-acting elements contribute to a transcriptional enhancer activity found in the region between nucleotides-553 and -86 relative to the cap site. The enhancer was active in lymphoid DG75 cells but not in HeLa cells and stimulated transcription also from the heterologous thymidine kinase (TK) and beta-globin promoters. Nuclear extracts of lymphoid cells contained protein factors that bound to the enhancer. The in vitro introduction of a mutation in the enhancer sequence that substantially reduced the transcription stimulatory activity concurrently blocked the binding of one of the factors.

Role of serotonin in the regulation of interferon-gamma production by human natural killer cells.

Monocytes, recovered directly from peripheral blood by counter-current centrifugal elutriation (CCE), were shown to provide two regulatory signals for induction of interferon-gamma (IFN-gamma) in natural killer (NK) cells in response to interleukin-2 (IL-2): an upregulating signal and an inhibitory signal. The inhibitory signal was time-dependent, irreversible, and operating on a pretranslational level, as indicated by the inability of enriched NK cells to accumulate IFN-gamma mRNA in the presence of elutriated monocytes. Monocyte-induced inhibition of IFN-gamma production was abrogated by the biogenic amine serotonin, acting via the 5-hydroxytryptamine, or serotonin (5-HT1A), subset of serotonin receptors (5-HTR). Thereby, serotonin effectively promoted IFN-gamma production in the presence of monocytes. We conclude that serotonergic 5-HT1A receptors transduce signals that are required for NK cells to produce IFN-gamma in response to IL-2.

Angiotensin-converting enzyme gene I/D polymorphism in malignant hypertension.

BACKGROUND: The mechanism of the rapid transition of a stable benign hypertensive disease to a severe and devastating malignant hypertension is not fully understood. However, the renin angiotensin system, which is highly activated in malignant hypertension, is established as an important pathogenetic factor in different cardiovascular and renal diseases. Over the last decade, a polymorphism in genes regulating this system has been found. This includes the 287 bp sequence deletion (D)/insertion (I) polymorphism in the angiotensin-converting enzyme (ACE) gene and the methionine (M) to threonine (T) point mutation polymorphism in the angiotensinogen (AGT) gene. These gene polymorphisms have been associated with various cardiovascular and renal diseases and the aim of this study was to investigate whether they were linked to malignant hypertension. METHODS: Forty-two patients with malignant hypertension (mean age 55 years), 42 patients with non-malignant hypertension (mean age 57 years) and 85 normotensive control subjects (mean age 42 years) were investigated with respect to ACE I/D and AGT M/T genotypes. DNA was prepared by standard methods from isolated white blood cells and analysed by the PCR technique. The PCR reaction used in the detection of the ACE I/D polymorphism was optimized for an equal amplification of the I and D alleles. RESULTS: The frequency of the DD genotype was significantly increased in patients with malignant hypertension (43%) compared with patients with non-malignant hypertension (14%) and normotensive control subjects (18%) (p <0.01) for both. The frequency distribution of AGT M/T genotype did not differ between patients with malignant and non-malignant hypertension. CONCLUSION: The DD genotype of the ACE gene occurred more than twice as often in malignant hypertension than in non-malignant hypertension and indicates that ACE gene polymorphism is a significant risk factor for initiation of malignant hypertension.

Cytomegalovirus infection of the CNS in non-compromised patients.

Six non-compromised patients with cytomegalovirus (CMV) associated meningoencephalitis are described. CMV was isolated from the cerebrospinal fluid (CSF) in 2/4 cases, while the diagnosis was based on an 8-fold rise in CMV-specific serum IgG antibodies and intrathecal antibody production against CMV in one case. By the polymerase chain reaction (PCR) CMV DNA was detected in the CSF in 5/5 cases and in serum in 3/4 cases. In one patient who had an Influenza A infection, both CMV and Epstein-Barr virus DNA were detected by PCR in the CSF. In 4 patients possible triggering events could be identified. Symptoms and signs indicating a multifocal brain involvement were present in 4 patients. The outcome was generally favourable except for sequelae in form of slight dysphasia in one case.

Polymerase chain reaction for monitoring human papillomavirus contamination of medical personnel during treatment of genital warts with CO2 laser and electrocoagulation.

Genital warts and intraepithelial neoplasia caused by infection with human papillomavirus are usually treated with CO2 laser or electrocoagulation. In this study, contamination of personnel and operating theatres with human papillomavirus DNA during treatment sessions was investigated. Samples were taken from the nostrils, nasolabial folds and conjunctiva of the operating physician before and after operating sessions and from Petri dishes left open in the operating theatres. Human papillomavirus DNA was demonstrated by the polymerase chain reaction technique. The results show that there is a risk of contamination of the operator by human papillomavirus DNA, detectable with the polymerase chain reaction technique, during both CO2 laser and electrocoagulation treatment.

Detection of cytomegalovirus DNA in serum correlates with clinical cytomegalovirus retinitis in AIDS.

The high sensitivity of nested polymerase chain reaction (PCR) offers the possibility of rapid detection of cytomegalovirus (CMV) DNA in serum. Five consecutive serum samples were examined from 52 human immunodeficiency virus (HIV)-seropositive patients (19 of whom had clinically presumed diagnosis of CMV chorioretinitis). Presence of CMV DNA in serum was shown to precede development of clinical disease. Eleven patients who developed chorioretinitis were positive for CMV DNA in serum samples obtained 3 months before clinical disease, and 3 retinitis patients who initially were negative for CMV DNA became positive with the onset of clinical retinitis. In contrast, 29 of 33 HIV-seropositive subjects without clinical CMV chorioretinitis and matched with respect to age and CD4 T cell numbers were negative for CMV DNA in all 5 serum samples. Thus, the presence of CMV DNA in serum analyzed by PCR is a good predictive marker of CMV retinitis in HIV-seropositive subjects. A positive PCR results supports the clinical diagnosis and may be useful for monitoring response to antiviral treatment.

Clinical evaluation in organ transplant patients of a polymerase chain reaction test for CMV DNA applied on white blood cells and serum.

A polymerase chain reaction (PCR) test for CMV DNA was evaluated for clinical usefulness. Leukocytes and serum were sampled from 36 patients who had recently undergone organ transplantation. Clinical symptoms, virus culture, and IgG and IgM antibodies were used to identify, in retrospect, patients with CMV disease certified beyond all doubt, with probable disease, with asymptomatic infection, or without infection. PCR tests for CMV DNA in leukocytes (BC-PCR) and serum (SE-PCR) were then evaluated. BC-PCR was positive in all patients with certified CMV disease but also in 31% of the samples from patients without infection. SE-PCR was positive in 11/13 patients with certified disease and was concordant with CMV culture in 192/231 tests. Of the 39 discordant cases, 27 had a positive SE-PCR with a negative culture. The effect of ganciclovir treatment could not be predicted by any test. In conclusion, a negative BC-PCR is strong evidence against CMV disease and a positive SE-PCR strongly suggests CMV disease, but the opposite results are of little clinical help.