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.

No association between the alpha2-macroglobulin (A2M) deletion and Alzheimer's disease, and no change in A2M mRNA, protein, or protein expression.

A polymorphism consisting of a deletion near the 5' splice site of exon 18 on the alpha2-macroglobulin (A2M) gene (A2M-2) has been suggested to be associated with Alzheimer's disease (AD) in family-based studies. We studied the A2M-2 allele together with the ApoE alleles in a large series on patients with AD (n = 449) and age-matched controls (n = 349). Neuropathologically confirmed diagnoses were available in 199 cases (94 AD and 107 control cases). We found no increase in A2M-2 genotype or allele frequencies in AD (27.5% and 14.6%) versus controls (26.4% and 14.9%). In contrast, a marked increase (p < 0.0001) in ApoE epsilon4 genotype or allele frequencies was found in AD (66.6% and 41.2%) as compared with controls (29.8% and 16.5%), suggesting sufficient statistical power in our sample. No relation was found between the A2M-2 and the ApoE epsilon4 allele. No change in A2M exon 17-18 mRNA size or sequence or A2M protein size was found in cases carrying the A2M-2 deletion, suggesting that there is no biological consequences of the A2M intronic deletion. No change in A2M protein level in cerebrospinal fluid was found in AD, suggesting that the A2M-2 allele does not effect the A2M protein expression in the brain. The lack of an association between the A2M-2 allele and AD in the present study, and the lack of abnormalities in the A2M mRNA or protein suggest that the A2M-2 allele is not associated with AD.

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.

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.

Angiotensin I-converting enzyme gene polymorphism in non-diabetic renal disease.

BACKGROUND: The insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene determines the concentration of ACE in serum and local tissues. The role of this polymorphism in progressive chronic renal disease is still not fully clear. METHODS: We analysed the impact of the D/D polymorphism on the rate of decline in renal function in patients with non-diabetic, chronic progressive renal insufficiency. Seventy non-diabetic patients, aged 21-69 years at baseline, with moderately advanced renal insufficiency due to primary chronic renal disease were followed for an average of 3 years with repeated measurements of their glomerular filtration rate (GFR). Their mean GFR at baseline was 41 ml/min/1.73 m(2) body surface area (BSA). The polymerase chain reaction (PCR) amplification method was used to detect the I/D polymorphism of the ACE gene. GFR was measured as the clearance of (51)Cr-EDTA and the individual rate of progression was calculated using linear regression. RESULTS: The distributions of the genotypes were: D/D 30%, I/D 49%, and I/I 21%. The rates of progression in the three ACE genotype groups were an annual decline in renal function of -4.2 (SD 4.6) ml/minx1.73 m(2) BSA in the D/D group, -2.7 (SD 3. 4) in the I/D group and -1.7 (SD 3.4) in the I/I group (ANOVA P=0. 12). In patients with proteinuria below 3.5 g/24 h, the D/D group had a significantly higher rate of progression than patients with the I allele. The same was found in a separate analysis when only patients with normal apoliprotein B (below 155 mg/dl) levels were analysed. Furthermore, the D/D genotype was a significant predictor of a more rapid decline in renal function in male, but not female, patients. CONCLUSION: The results in this study in non-diabetic patients with chronic renal disease indicate that the presence of the D allele in the ACE genotype may be of particular importance as a predictor of a high rate of progression in male patients who otherwise do not have a major burden of documented and important prognostic factors for progressive renal insufficiency.

Relative levels of EBNA1 gene transcripts from the C/W, F and Q promoters in Epstein-Barr virus-transformed lymphoid cells in latent and lytic stages of infection.

Four promoters, Cp, Wp, Fp and Qp, are known to participate in transcription of the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) gene in EBV-infected cell lines. The promoters are used differentially during the different phases of infection and establishment of the stages of latency. This has raised questions about the regulation of the promoters and the molecular mechanisms underlying the switches between them. To obtain a measure of the activity of the different EBNA1 transcription units in EBV-transformed cell lines of different phenotypes, RNA probes were constructed that allowed the detection and relative quantification, by RNase protection analysis, of EBNA1 transcripts initiated at Fp and Qp and, in an indirect manner, Cp/Wp. RNase protection and PCR assays were performed with cytoplasmic RNA from B-lymphoid cell lines in latency stages I, II-III and III and after induction of the virus lytic cycle. The experiments demonstrated that, in addition to previously identified EBNA1 transcripts, cell lines of all latency types also contained different mRNAs that carried sequences from the EBNA1-encoding K exon. Induction of the virus lytic cycle resulted in low levels of an FpQ/U/K-spliced transcript. However, there was a large increase of FpQ- and FpQ/U-spliced transcripts with unknown 3' sequences. Furthermore, a new transcript, initiated at an unidentified site 5' of the BamHI f/K cleavage site and continuing through BamHI K into the EBNA1-encoding K exon without interruption, was produced in substantial amounts in the lytic cycle.

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.

Congenital toxoplasma gondii infection diagnosed by PCR amplification of peripheral mononuclear blood cells from a child and mother.

A case of congenital toxoplasmosis is presented, where diagnosis by PCR amplification of Toxoplasma gondii DNA from peripheral blood led to early treatment of the infant and seemingly normal brain development despite the presence of intracranial calcifications at birth. The mother, who experienced a subclinical infection during pregnancy, was PCR-positive for toxoplasma DNA in a sample of peripheral blood drawn after delivery.

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.

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.

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.

Conserved N-linked oligosaccharides of the C-terminal portion of human immunodeficiency virus type 1 gp120 and viral susceptibility to neutralizing antibodies.

We have constructed a mutated infectious HIV variant lacking the signals for addition of three N-linked glycans situated in the V4, C4 and V5 regions of HIV gp120. When comparing mutated virus with wildtype virus we found essentially no differences in the phenotypic characteristics of the two viruses except for the expected electrophoretic mobility shift of radioimmuno-precipitated mutated gp120, resulting from the missing N-glycans. Thus, the infectivity titer and the capacity to induce syncytia were similar for the two viruses. The sensitivity of mutant and wildtype virus to a number of neutralizing agents was determined. As expected, the mutant virus was significantly less sensitive to neutralization by Con A, with affinity for the N-glycans eliminated. We found, however, that antibodies to the V3 loop and sCD4 neutralized wild-type virus as efficiently as mutant virus, whereas 2G12, a monoclonal antibody, binding to a discontinuous neutralization epitope, and GP13, binding to the CD4-binding domain, neutralized wildtype virus better than mutant virus. Altogether the data suggest that the three conserved N-linked glycans, despite their location in immediate association with the CD4-binding domain, which is an important neutralization epitope, are not essential for virus replication in cell culture and they are not engaged in shielding neutralization epitopes of gp120 from neutralizing antibodies. However, the glycans evidently influence the three-dimensional conformation of gp120, since their presence increases the availability of the neutralization epitope of 2G12.

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.

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.

[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.

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.

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.

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.

Diagnosis of Epstein-Barr virus-induced central nervous system infections by DNA amplification from cerebrospinal fluid.

A nested polymerase chain reaction was used for the detection of Epstein-Barr virus DNA in 1 patient with encephalitis, and in 1 patient with myelitis. Epstein-Barr virus DNA was detected in cerebrospinal fluid samples obtained at the onset of neurological symptoms in both patients, and serological findings indicated ongoing Epstein-Barr virus infection. In the patient with encephalitis, herpes simplex virus type 1 DNA was transiently detected in the cerebrospinal fluid, while Epstein-Barr virus DNA was still present on day 44 after admittance. Single-photon emission computed tomography in this patient indicated a frontal bilateral hypoperfusion. The diagnostic value of polymerase chain reaction on cerebrospinal fluid and serum samples for Epstein-Barr virus infections of the central nervous system is emphasized.