Separate Polymerase Chain Reaction Research Articles

Analysis of Vaginal Swabs for Paternity Testing and Marker-Assisted Selection in Cattle

Blood is the standard source for DNA analysis, but requires venipuncture of cows by veterinarian and tedious and costly DNA extraction. A procedure was developed for sampling of vaginal cells from cows, establishing a cell lysis protocol using robotics, and applying fluorescent analysis of genetic markers. Two insemination technicians collected vaginal cells from 254 elite Israeli Holstein cows located in 152 herds using commercial Catch-All sample collection brushes. Cells were lysed in a 400-μl solution, and 5μl was used as template for polymerase chain reaction (PCR). Sensitivity of the PCR was enhanced using only 1μl of lysed cells. Eight markers of the International Society of Animal Genetics paternity panel were amplified in four separate PCR. ILSTS039, a marker for a quantitative trait loci on BTA14, was amplified in a separate reaction. Genotypes from one to nine genetic markers were obtained for 253 out of 254 samples, and 244 cows had genotypes for at least three markers (96%). Cows that did not inherit either paternal allele for at least two loci were considered not to be daughters of the sire listed. Fifteen cows met this criterion, for a paternity misidentification rate of 6.25%. The frequency of allele 225 of ILSTS039, which was associated with increased milk fat content, was 11.1% in the bull-dam population, similar to the 12% found in the cow population in Israel. The use of vaginal cells for genetic analysis is accurate, as demonstrated by replicated analysis and by comparison to individual and population analysis based on DNA derived from blood.

A Molecular Variant of Angiotensinogen Is Associated With Idiopathic Intrauterine Growth Restriction

In Brief OBJECTIVE Intrauterine growth restriction has been associated with failed maternal physiologic changes such as abnormal spiral artery remodeling and reduced maternal blood volume. A polymorphism of angiotensinogen Thr235 has been considered a risk factor for preeclampsia. We genotyped maternal and fetal deoxyribonucleic acid (DNA) for angiotensinogen Thr235 to estimate whether the polymorphism is also a risk factor for intrauterine growth restriction. METHODS We examined maternal blood DNA in 174 patients with intrauterine growth restriction and 60 patients with both preeclampsia and intrauterine growth restriction. The control group comprised 400 consecutive cases of women with term pregnancies and infants with birth weight between the fifth and 95th percentiles. We also examined 162 DNA samples from fetal blood with intrauterine growth restriction for the Thr235 polymorphism, and 240 normal fetuses were used as the control group. The angiotensinogen genotype was determined using mutagenically separated polymerase chain reaction. The products were size fractionated on an agarose gel. Angiotensinogen genotypes were divided into three groups: MM (homozygous for angiotensinogen Met235 allele), TT (homozygous for angiotensinogen Thr235 allele), and MT (heterozygous). RESULTS Maternal genotyping revealed a significantly higher Thr235 allele frequency in intrauterine growth restriction (.60) and preeclampsia/intrauterine growth restriction (.63) than in the control group (.36) (P < .001). Fetal genotyping revealed a Thr235 allele frequency of .59 in intrauterine growth restriction fetuses, as compared with the control group (.38) (P < .001). CONCLUSION Maternal and fetal angiotensinogen Thr235 genotypes are associated with an increased risk of intrauterine growth restriction in our study population. The angiotensinogen Thr235 allele may predispose women to deliver growth-restricted fetuses. Women with the angiotensinogen Thr235 mutation are at increased risk for intrauterine growth restriction and pregnancy-induced hypertension.

A molecular variant of angiotensinogen is associated with idiopathic intrauterine growth restriction

Objective Intrauterine growth restriction has been associated with failed maternal physiologic changes such as abnormal spiral artery remodeling and reduced maternal blood volume. A polymorphism of angiotensinogen Thr235 has been considered a risk factor for preeclampsia. We genotyped maternal and fetal deoxyribonucleic acid (DNA) for angiotensinogen Thr235 to estimate whether the polymorphism is also a risk factor for intrauterine growth restriction. Methods We examined maternal blood DNA in 174 patients with intrauterine growth restriction and 60 patients with both preeclampsia and intrauterine growth restriction. The control group comprised 400 consecutive cases of women with term pregnancies and infants with birth weight between the fifth and 95th percentiles. We also examined 162 DNA samples from fetal blood with intrauterine growth restriction for the Thr235 polymorphism, and 240 normal fetuses were used as the control group. The angiotensinogen genotype was determined using mutagenically separated polymerase chain reaction. The products were size fractionated on an agarose gel. Angiotensinogen genotypes were divided into three groups: MM (homozygous for angiotensinogen Met235 allele), TT (homozygous for angiotensinogen Thr235 allele), and MT (heterozygous). Results Maternal genotyping revealed a significantly higher Thr235 allele frequency in intrauterine growth restriction (.60) and preeclampsia/intrauterine growth restriction (.63) than in the control group (.36) ( P < .001). Fetal genotyping revealed a Thr235 allele frequency of .59 in intrauterine growth restriction fetuses, as compared with the control group (.38) ( P < .001). Conclusion Maternal and fetal angiotensinogen Thr235 genotypes are associated with an increased risk of intrauterine growth restriction in our study population. The angiotensinogen Thr235 allele may predispose women to deliver growth-restricted fetuses.

The transforming growth factor-β1 gene polymorphism (G915C) is not associated with systemic lupus erythematosus

Lymphocyte production of transforming growth factor (TGF)-beta1 is decreased in systemic lupus erythematosus (SLE). The lack of this immunoregulatory cytokine may contribute to the characteristic T cell disregulation and aberrant B cell stimulation in SLE patients. The less common C allele of the TGFB1 polymorphism (G915C) is associated with a lower TGF-beta1 production capacity. We performed a population-based case-control study to analyse the impact of this polymorphism on disease susceptibility, on clinical SLE manifestations and autoantibody production. A total of 203 German Caucasian SLE patients (fulfilling the 1982 ACR disease duration 11.5 +/- 7.0 years) and 158 ethnically, age- and sex-matched healthy controls were genotyped with a mutagenically separated polymerase chain reaction. There were no significant differences in the genotype distribution and allele frequencies between patients (915 C = 0.08) and healthy controls (915 C = 0.10). Comparing subgroups of patients, we found no association of major disease manifestations or specific autoantibodies with TGFB1 genotypes or alleles. The TGFB1 polymorphism (G915C) neither significantly contributes to the disease susceptibility, nor predisposes to clinical and immunological manifestations typical of SLE. Further studies are needed to corroborate the pathogenic role of TGF-beta1 in SLE patients and to identify the precise genetic elements controlling its production.

2P-0415 Rapid detection of uncoupling protein 2-866G/A mutation by mutagenically separated polymerase chain reaction (MS-PCR)

2P-0415 Rapid detection of uncoupling protein 2-866G/A mutation by mutagenically separated polymerase chain reaction (MS-PCR)

High prevalence of Neisseria gonorrhoeae and multiple sexually transmitted diseases among rural women in the Eastern Highlands Province of Papua New Guinea, detected by polymerase chain reaction.

In a previous community-based study among rural women in the Eastern Highlands Province (EHP) of Papua New Guinea we determined that the prevalences of Trichomonas vaginalis infection, Chlamydia trachomatis infection, and syphilis were 46%, 26%, and 4%, respectively. Surprisingly, however, the prevalence of Neisseria gonorrhoeae infection was only 1%, which we considered low in consideration of the high prevalence of other sexually transmitted diseases (STDs). The aim of the current study was to reexamine samples that were collected in that survey and retest them with use of polymerase chain reaction (PCR). Using a cluster-sampling method, we surveyed 201 women aged 15 to 45 years in a population of approximately 19,000 people. In addition, 243 other women living in the same area who wished to be screened for STDs were included in the study. Endocervical samples that were stored frozen at -80 degrees C were retested with multiplex PCR (M-PCR) for the detection of both N gonorrhoeae and C trachomatis and with a separate PCR for the detection of T vaginalis. A total of 373 samples that were still available were analyzed. The prevalences of T vaginalis, C trachomatis, and N gonorrhoeae infections were 42.6%, 26.5%, and 18.2%, respectively; 59.8% of the women had at least one STD, while 21.7% had mixed infections, 5.9% of them with all three pathogens. STDs are very common among rural women in the EHP of Papua New Guinea and often present as multiple infections.

The mutagenically separated polymerase chain reaction is a rapid and reliable method for genotyping of the tumour necrosis factor-α promoter polymorphism (−308 G/A)

Background: The tumour necrosis factor-α (TNFα) promoter polymorphism (−308 G/A) has been shown to be associated with the susceptibility to and/or the severity of diverse diseases such as infections, autoimmunity, and malignancies. We developed a genotyping technique based on the mutagenically separated polymerase chain reaction (MS-PCR) which may be useful in the clinical risk assessment. Methods: Different length allele-specific primers and an unspecific complementary strand primer were used in a one-tube assay. At least one PCR product was generated in a single reaction obviating the need for an internal control amplification. Introduction of additional base substitutions into the allele-specific primers led to a clear-cut separation between the alleles through the reduction of cross-reactions during amplification. The only post-PCR step required was the separation of allelic PCR products by size upon agarose gel electrophoresis. Results: The allele frequencies in 300 German healthy Caucasians were 0.84 for TNF1 (−308 G) and 0.16 for TNF2 (−308 A) in accordance with published data obtained with the conventional RFLP method. No significant deviation from Hardy–Weinberg equilibrium was observed. The specificity of MS-PCR was confirmed by sequence-based typing. Conclusions: MS-PCR is a rapid, reliable, and cost-effective technique for genotyping of the TNFα promoter polymorphism (−308 G/A).

Studying the heterogeneity of brain tumors using medium throughput LOH analysis.

The theory of tumor progression by clonal evolutionposits that a succession of increasingly malignant clonesarise, in turn, driven by the step-wise accumulation ofmutations (1). At each point in this process it is thoughtthat one cell in the currently dominant clone acquires anew mutation, allowing it to outcompete its siblings. As aresult, its descendants enjoy a higher rate of net growth,making them the new dominant clone whose growtheclipses that of preceding clones, until it too is left behindin the wake of the next mutation. Some direct evidencefor this model exists, for example, from analyzing the roleof TP53 in brain tumors. In an elegant study of primaryand recurrent glioma, it was possible to find the mutationprevalent in recurrent tumors represented in some cells ofthe primary lesion, suggesting that this molecular defectmarked the dominant clone about to emerge (2).In the strictest interpretation of the clonal evolutionmodel, a single clone makes up the bulk of the tumor at agiven stage (Fig. 1a). Any observed tumor heterogeneity isconsidered to be due to the remains of ancestral clones,whose sidelined descendants may persist but do not de-fine the clinical course of the disease. This view fits thedata from many studies where tumors are considered tobe, to a first approximation, homogeneous entities, and allof a tumor’s cells are combined in one sample and ana-lyzed using molecular markers of progression. This inter-pretation also is reassuring in its implication that effectivetreatment will result from targeting the dominant clone.The validity of the inherent assumptions that a tumor canbe treated as a single entity is an increasingly importantconcern, particularly as new therapies based on the per-ceived biological characteristics of an individual patient’stumor, become available.Although what may be termed the dominant cloneinterpretation of the theory of clonal evolution may verywell describe many tumor types, particularly those char-acterized by extreme changes in growth rate and invasive-ness with concomitant well-defined histologic stages, itmay not explain all cancers. Here we discuss data fromanalyses of loss of heterozygosity (LOH), which measuresclonally inherited genetic damage associated with the in-activation of tumor suppressor genes (3). Therefore LOHrelates directly to cancer progression and also serves as atool for following clonal patterns. Our data suggest thatother views of the theory of clonal evolution besides thedominant clone interpretation may need to be consideredin some tumor types.

Cardiovascular phenotype of young adults and angiotensinogen alleles.

Angiotensinogen (AGT) gene variants influence angiotensinogen plasma levels in children and young adults. The angiotensinogen promoter (-6)A variant facilitates gene transcription in human tissues and it has been associated with high blood pressure in older adults. A young adult population can be used as a model to study genotype/phenotype associations between AGT (-6) variants and cardiovascular variables. Anthropometric measurements, blood pressure and heart rate were taken in 422 white Caucasian students (mean age 23.5 years, SD 2.5 years). Family history for hypertension, physical activity and smoking history were evaluated. Left ventricular variables were measured by echocardiography. Carotid artery wall intimal-media thickness (IMT) was measured by high resolution sonography and digitalized morphometry. The AGT G(-6)A alleles were evaluated by mutagenically separated polymerase chain reaction controlled by direct sequencing. No significant associations were found between angiotensinogen genotype and blood pressure, cardiac variables [except for deceleration time in females which increased with the number of (-6)A alleles] and IMT. Allele frequencies were similar between the first and third tertile of blood pressure and left ventricular mass, and were also similar between negative or positive family history for hypertension (the last group having significantly higher systolic blood pressure in males, P = 0.04 and diastolic blood pressure in females, P < 0.01). Moreover, no relevant interaction on the cardiovascular variables was found between AGT genotype and body mass index. The angiotensinogen G(-6)A variants do not affect cardiovascular parameters in young adults, but an effect of this polymorphism on cardiovascular phenotype (and hypertension) in older adults cannot be excluded. Additional factors, associated with ageing, should be present to unleash the supposed unfavourable potential of the (-6)A angiotensinogen variant.

Association between the α-adducin Gly460Trp polymorphism and systolic blood pressure in familial combined hyperlipidemia

Background In a genome scan for familial combined hyperlipidemia (FCHL), a locus contributing to systolic blood pressure (SBP) has been identified on chromosome 4, containing the α-adducin gene ( ADD1). In previous studies, an association has been found between the α-adducin Gly460Trp polymorphism and salt-sensitive hypertension. In this study, we investigated the association between the α-adducin Gly460Trp polymorphism and blood pressure in FCHL patients. Methods A total of 79 unrelated patients with FCHL and 121 unrelated controls (spouses) were recruited for the study. Blood pressure was measured in a standardized fashion, with the subject in sitting position after 10 min of rest. The α-adducin Gly460Trp polymorphism was detected by mutagenically separated polymerase chain reaction. Results The genotype frequencies of both FCHL patients and controls were in Hardy-Weinberg equilibrium. The α-adducin Gly460Trp polymorphism showed a significant association with FCHL, the number of subjects carrying a 460Trp allele was significantly higher in patients compared with controls (53% v 33%, χ 2 = 8.0, P = .018). In FCHL patients carrying at least one 460Trp allele, SBP was significantly higher compared with patients homozygous for the 460Gly allele (140 mm Hg and 130 mm Hg respectively, P = .015). Conclusions This study shows that the 460Trp allele is associated with FCHL. Furthermore, SBP is increased in patients carrying the 460Trp allele.

Molecular Identification and Classification of a Phytoplasma Associated with Phyllody of Strawberry Fruit in Maryland.

Several phytoplasmas have been reported to be associated with phyllody of strawberry fruit, including clover yellow edge, clover proliferation, clover phyllody, eastern and western aster yellows, STRAWB2, strawberry multicipita, and Mexican periwinkle virescence phytoplasmas. Plant symptoms in addition to phyllody may include chlorosis, virescence, stunting, or crown proliferation. In this report we describe a new phytoplasma in association with strawberry leafy fruit (SLF) disease in Maryland. Diseased plants exhibited fruit phyllody, floral virescence, leaf chlorosis, and plant stunting. Phytoplasmal 16S rDNA was amplified from SLF diseased plants by using the polymerase chain reaction (PCR) primed by primer pair P1/P7 and was reamplified in nested PCR primed by primer pair R16F2n/R2 (F2n/R2) as previously described (1). These results indicated the presence of a phytoplasma, designated SLF phytoplasma. Identification of SLF phytoplasma was accomplished by restriction fragment length polymorphism (RFLP) analysis of DNA amplified in PCR primed by F2n/R2, using endonuclease enzyme digestion with AluI, HhaI, KpnI, HaeIII, MseI, HpaII, RsaI, and Sau3AI. Phytoplasma classification was done according to the system of Lee et al. (2). RFLP analyses of rDNA amplified in three separate PCRs gave identical patterns. On the basis of collective RFLP patterns of the amplified 16S rDNA, the SLF phytoplasma was classified as a member of group 16SrIII (group III, X-disease phytoplasma group). The HhaI RFLP pattern of SLF 16S rDNA differed from that of the apparently close relative, clover yellow edge (CYE) phytoplasma, and all other phytoplasmas previously described in group III. Based on these results, SLF phytoplasma was classified in a new subgroup, designated subgroup K (III-K), within group III. The 1.2 kbp DNA product of PCR primed by primer pair F2n/R2 was sequenced, and the sequence deposited in GenBank under Accession No. AF 274876. Results from putative restriction site analysis of the sequence were in agreement with the results from actual enzymatic RFLP analysis of rDNA amplified from phylloid strawberry fruit. Although the sequence similarity between the 1.2-kbp fragment from the 16S rDNA of SLF phytoplasma and that of CYE phytoplasma was 99.9%, the Hha1 RFLP pattern of SLF rDNA supports the conclusion that the SLF phytoplasma may be closely related to, but is distinct from, CYE and other strains that are classified in group III. These findings contribute knowledge about the diversity of phytoplasmas affiliated with group III and the diversity of phytoplasmas associated with diseases in strawberry.

A Common C→T Polymorphism at nt 46 in the Promoter Region of Coagulation Factor XII is Associated With Decreased Factor XII Activity

Coagulation factor XII (FXII) deficiency is rarely found to be associated with bleeding, but single reports demonstrated thromboembolic events in FXII-deficient patients. Currently, the biological role of FXII is still discussed controversially. It is well known that plasma levels of FXII show great interindividual variability. Recently, it has been demonstrated that a frequently occurring C→T polymorphism in the FXII promoter region at nucleotide (nt) 46 is associated with lower plasma FXII activity levels in Orientals. In our study, we evaluated the frequency of this polymorphism in a randomly selected sample of newborns and investigated whether this C→T polymorphism also contributes to the frequently observed moderate FXII deficiency in Europeans. We developed a new mutagenically separated polymerase chain reaction assay (MS PCR), which allows mutation detection without the use of restriction enzymes. Among 100 healthy newborns, we found 64% homozygous carriers of the wildtype FXII 46C allele, 29% were heterozygous for FXII C46T, and 7% homozygous for FXII 46T. Evaluation of plasma FXII activity and genotype in 80 randomly selected and unrelated individuals revealed a highly statistically significant ( P<.001) association of the FXII 46T allele with reduced FXII plasma activity. Individuals carrying the homozygous FXII 46C genotype had a mean of 1.17 U/ml (±0.31 U/ml), individuals heterozygous for FXII C46T showed a mean of 0.70 U/ml (±0.31 U/ml), and subjects homozygous for FXII 46T had only 0.44 U/ml (±0.10 U/ml) plasma FXII activity.

Herpes Simplex Virus DNA in Amniotic Fluid without Neonatal Infection

Twenty-one pregnant women were studied to determine the distribution of herpes simplex virus (HSV): 10 had symptomatic genital herpes, including 1 with primary cervical HSV infection, and 11 had asymptomatic genital herpes. Samples from vesicles, the cervix, and amniotic fluid (AF) were analyzed with 2 separate polymerase chain reaction (PCR) tests and with viral culture. For newborns, clinical examinations and pharyngeal HSV cultures were performed, and cord blood IgM antibodies to HSV were measured. HSV DNA was present in the AF of 3 women with symptomatic HSV infection, but all cultures were negative. HSV was detected more often with PCR than with culture, in both vesicle and cervical samples. For the asymptomatic group, all AF samples were negative, whereas 4 cervical samples were positive by PCR (none were positive by culture). All isolates were HSV type 2. All infants were healthy, and none had cord blood IgM antibodies to HSV, including those with PCR-positive AF.

Detection of Microsatellite Instability by Fluorescence Multiplex Polymerase Chain Reaction

We have created a clinical molecular diagnostic assay to test for microsatellite instability (MSI) at multiple loci simultaneously in paraffin-embedded surgical pathology colon resection specimens. This fluorescent multiplex polymerase chain reaction (PCR) assay analyzes the five primary microsatellite loci recommended at the 1997 National Cancer Institute-sponsored conference on MSI for the identification of MSI or replication errors in colorectal cancer: Bat-25, Bat-26, D2S123, D5S346, and D17S250. Amplicon detection is accomplished by capillary electrophoresis using the ABI 310 Genetic Analyzer. Assay validation compared 18 specimens previously assessed by radioactive PCR and polyacrylamide gel electrophoresis detection to results generated by the reported assay. Germline and tumor DNA samples were amplified in separate multiplex PCR reactions, sized in separate capillary electrophoresis runs, and compared directly to identify novel length alleles in tumor tissue. A concordance of 100% between the two modalities was achieved. The multiplex assay routinely detected a subpopulation of 10% tumor alleles in the presence of 90% normal alleles. A novel statistical model was generated that corroborates the validity of using results generated by analysis of five independent microsatellites to achieve a single overall MSI diagnosis. The assay presented is superior to standard radioactive monoplex PCR, polyacrylamide gel electrophoretic analysis, primarily due to the multiplex PCR format.

Detection and identification of the maize bushy stunt phytoplasma in corn plants in Brazil using PCR and RFLP

Plants of corn (Zea mays L.) exhibiting symptoms of stunting and leaf reddening were assayed for the presence of phytoplasma gene sequences through the use of phytoplasma rRNA and ribosomal protein gene and maize bushy stunt (MBS) phytoplasma-specific oligonucleotide primers in polymerase chain reactions (PCR). Polymorphisms in 16S rDNA amplified from diseased plants were those characteristic of phytoplasmas classified in the16S rRNA gene group 16SrI, subgroup IB, of which MBS phytoplasma is a member. Amplification of ribosomal protein (rp) gene sequences in PCR primed by phytoplasma-specific primers confirmed presence of a phytoplasma in the diseased plants. Restriction fragment length polymorphism (RFLP) patterns of the amplified phytoplasma rp gene sequences were similar or identical to those observed for a known strain of MBS phytoplasma. In separate PCR, an MBS-specific oligonucleotide pair primed amplification of a MBS-characteristic DNA from templates derived from the diseased corn. Our data provide the first firm evidence for the presence of maize bushy stunt phytoplasma in corn in Brazil.

Analysis of five Duchenne muscular dystrophy exons and gender determination using conventional duplex polymerase chain reaction on single cells.

We have developed five conventional duplex polymerase chain reaction (PCR) protocols on single lymphocytes and blastomeres from embryos, in order to analyse five exons commonly deleted in deletion-type Duchenne muscular dystrophy (DMD). The five DMD gene exons (17, 19, 44, 45 and 48) can be analysed in separate duplex PCR reactions together with the sex-determining region Y (SRY) gene which enables simultaneous gender assignment. We present here PCR amplification results from single lymphocytes isolated from a normal male (220 cells), a normal female (24 cells) and a male DMD patient (40 cells) carrying a deletion of exons 46-49 within the DMD gene. The method failed to produce a PCR signal for the SRY gene in 8/220 normal male cells (3.6%) and for a DMD exon in 0-4.5% of normal male cells. One negative control out of 112 was positive. When this method was used to analyse two blastomeres from each of five embryos, concordant results were obtained for each pair of blastomeres. All embryos produced signals for the DMD exon tested with four of the embryos found to be male and one female. This method is therefore suitable for preimplantation genetic diagnosis and will allow the transfer of healthy embryos (both male and female) in families carrying DMD gene deletions involving at least one of the five exons 17, 19, 44, 45 and 48.

A(-6)G variant of angiotensinogen gene and aldosterone levels in hypertensives.

Recently, a novel mutation in the promoter region of the angiotensinogen gene that involves the presence of an adenine instead of a guanine 6 bp upstream from the transcription initiation site (A(-6)G) has been shown to induce an increase in gene transcription. The aim of this study was to determine the prevalence of the A(-6)G mutation in essential hypertensive patients and to correlate it with aldosterone and renin activity levels. We studied 191 hypertensives. We measured levels of aldosterone (plasma and urinary) and plasma renin activity. We determined the variants A and G using a mutagenically separated polymerase chain reaction technique. In 191 hypertensives, the A variant was detected in 266 of 382 (69.6%) and the G variant in 116 of 382 alleles (30.4%). Plasma aldosterone was significantly higher in patients homozygous for AA than in those homozygous for GG (369+/-208 versus 246+/-142 pmol/L). Urinary aldosterone was significantly higher in homozygous AA than in AG or GG patients (62.4+/-39.4 versus 50.8+/-25.2 and 37.4+/-22.3 nmol/d, respectively). When the patients were grouped according to the presence or absence of the A allele, the aldosterone levels and the plasma aldosterone/plasma renin activity ratio were significantly higher in patients with the A allele. The presence of the A variant was associated with higher levels of aldosterone. These results suggest that the presence of the A variant could determine the appearance of arterial hypertension through higher transcription activity of the angiotensinogen gene and concomitant aldosterone production.

A high-throughput MS-PCR method on MADGE gels for ANG II type-1 receptor A1166C polymorphism

We have developed a highly accurate, low-cost, single-step, mutagenically separated polymerase chain reaction (MS-PCR) method for the determination of angiotensin II type-1 receptor (AT(1)) A1166C gene polymorphism. The genotypes are determined using the microtiter array diagonal gel electrophoresis (MADGE) system. We have compared the MS-PCR method with allele-specific oligonucleotide hybridization and Dde I digestion techniques for determining the AT(1) A1166C genotype. The combination of MS-PCR and MADGE serves as a model for high-throughput single-nucleotide polymorphism genotyping in large population studies.

The role of parvovirus B19 in the pathogenesis of giant cell arteritis: a preliminary evaluation.

To determine whether parvovirus B19 DNA is more likely to be present in the temporal arteries of patients with giant cell arteritis (GCA) than in the temporal arteries of control subjects. We prospectively examined temporal artery biopsy (TAB) tissue from 50 consecutive patients presenting for TAB for the presence of B19 DNA using the polymerase chain reaction (PCR). Clinical and demographic information was obtained from the patients' medical records. A separate PCR analysis of 30 original tissue specimens was conducted at the Centers for Disease Control and Prevention (CDC) using primers directed toward another target sequence in the nonstructural coding area of B19. The 50 patients had an average age of 70.8 years; 27 (54%) were female. Amplicons for human beta-globulin, but not for cytomegalovirus, were produced for all tissue samples. The PCR results for B19 agreed in 29 of 30 samples tested by our institution and by the CDC (97% agreement; kappa = 0.9). A comparison of the B19 DNA analysis and the results of TAB indicated a statistically significant association between histologic evidence of GCA and the presence of B19 DNA in TAB tissue (chi2 = 10.38, P = 0.0013). These findings suggest that B19 may play a role in the pathogenesis of GCA.

Angiotensinogen Thr235 variant is associated with abnormal physiologic change of the uterine spiral arteries in first-trimester decidua

Objective: The angiotensinogen Thr235 polymorphism associated with preeclampsia is tightly linked to a mutation in the angiotensinogen promoter A(-6), which may lead to elevated expression in decidual spiral arteries. We hypothesize that locally elevated angiotensin II levels play a role in failed physiologic change leading to preeclampsia. Our objective was to determine whether spiral artery morphologic characteristics were different in first-trimester decidual samples from women homozygous for the angiotensinogen Thr235 allele and women homozygous for the normal angiotensinogen Met235 allele. Study Design: We used quantitative histologic analysis to study 1266 spiral artery cross-sections in decidual samples obtained from normal pregnancies (n = 53) terminated at 8 weeks' gestation. To define vessel characteristics before pregnancy-induced remodeling, we also examined 60 arteries in nonpregnant endometrial control samples (n = 5). We measured the aspect ratio, media area, and external diameter of each cross-section with Image-Pro plus software. Maternal angiotensinogen genotypes were determined by means of mutagenically separated polymerase chain reaction. Average spiral artery morphologic measurements were compared between genotypes with the Student t test. Results: The media area/external diameter ratio was lower in decidual samples than in endometrial samples ( P < .0001), consistent with pregnancy-induced physiologic changes. Women homozygous for the angiotensinogen Thr235 allele (n = 11) had a greater area/diameter ratio than did women homozygous for the normal angiotensinogen Met235 allele (n = 11, P < .05). Samples from heterozygous women (n = 31) had intermediate values. Conclusion: Our results suggest that the angiotensinogen Thr235 allele predisposes women toward abnormal physiologic change, potentially beginning the cascade of events leading to preeclampsia. (Am J Obstet Gynecol 1999;180:95-102.)