Heidi Buchmayer

Multiplex PCR for rapid detection of T cell receptor-gamma chain gene rearrangements in patients with lymphoproliferative diseases

We describe a multiplex polymerase chain reaction (PCR) method suitable for the detection of all T‐cell receptor (TCR) γ‐chain gene rearrangements in patients with lymphoproliferative diseases. 40 patients with various lymphoproliferative disorders and 40 healthy individuals were tested. Clonal TCRγ rearrangements were identified in all patients with malignant disorders, and in one of 10 cases with established reactive lymphocytosis but not in normal controls. In all individuals testing positive, the patient’s specific V and J segment involved in the rearrangement could be determined by simply splitting the multiplex primer mix. Our data show that the multiplex PCR technique enables rapid, simple and sensitive screening for clonal TCRγ chain gene rearrangements.

Efficacy of folinic versus folic acid for the correction of hyperhomocysteinemia in hemodialysis patients

The effectiveness of intravenous folinic acid or intravenous folic acid for the treatment of hyperhomocysteinemia of hemodialysis patients is unknown. In a randomized, controlled, double-blind trial, 66 hemodialysis patients were administered either 15 mg of folic acid or an equimolar amount (16.1 mg) of folinic acid intravenously three times weekly. Normalization of total homocysteine (tHcy) plasma levels after 4 weeks of treatment was achieved in 10 patients (30.3%) in the folic-acid group and 6 patients (18.2%; P: = 0.389) in the folinic-acid group (normalization at any time during the study period in 39.4% and 33.3% of the patients; P: = 0.798). The relative reduction in tHcy plasma levels at week 4 was 32.2% in the folic-acid group and 34.1% in the folinic-acid group. A high baseline tHcy plasma concentration (P: = 0.00001), methylenetetrahydrofolate reductase (MTHFR) 677TT/1298AA genotype (P: = 0.03540), and low red blood cell folate concentrations (P: = 0.02285) were associated with a better relative response to treatment. Normalization of tHcy plasma levels was dependent on a lower baseline tHcy level (P: = 0.01976), younger age (P: = 0.00896), and MTHFR 677TT/1298AA or 677CT/1298AC genotypes (P: = 0.00208 and P: = 0.02320, respectively). A 4-week course of intravenous folinic acid is not superior to intravenous folic acid in reducing elevated tHcy plasma levels in hemodialysis patients. The response to treatment is predicted by tHcy plasma level, red blood cell folate content, and MTHFR genotype.

Molecular Genetics of Homocysteine Metabolism

Recent genetic studies have led to the characterization of molecular determinants contributing to the pathogenesis of hyperhomocysteinemia. In this article we summarize the current insights into the molecular genetics of severe, moderate and mild hyperhomocysteinemia. We will consider deficiencies of the trans-sulfuration enzyme cystathionine β-synthase (gene symbol: CBS), and the disturbances of the remethylation enzymes 5,10-methylenetetrahydrofolate reductase (gene symbol: MTHFR), methionine synthase (gene symbol: MTR), and the recently identified methionine synthase reductase (gene symbol: MTRR). Furthermore, we will focus on clinically important genetic polymorphisms which are highly prevalent and thus of potential general interest.

High prevalence of hyperhomocysteinemia in critically ill patients.

Objective: To test the hypothesis that the prevalence of hyperhomocysteinemia is increased in critically ill patients and correlates with disease severity and mortality in these patients. Design: A prospective study. Setting: Three medical intensive care units at the University of Vienna Medical School serving both medical and surgical patients. Patients: All consecutive admissions (n = 56) during a period of 4 wks. A total of 112 age‐ and gender‐matched healthy individuals constituted the control group. Interventions: None. Measurements and Main Results: Blood samples were drawn within 24 hrs after admission for analysis of total homocysteine (tHcy), folate, vitamin B6 levels, and vitamin B12 levels as well as to identify the 677C→T polymorphism in the gene coding for the enzyme 5, 10‐methylenetetrahydrofolate reductase. Acute Physiology and Chronic Health Evaluation III scores at admission and 24 hrs after admission as well as 30‐day survival were documented in all patients. Hyperhomocysteinemia was more prevalent in critically ill patients (16.1%; 95% confidence interval, 7.6% to 28.3%) compared with age‐ and gender‐matched healthy individuals (5.4%; 95% confidence interval, 2.0% to 11.3%; chi‐square test; p = .022). There was no difference in tHcy plasma concentrations in the first 24 hrs after admission to an intensive care unit between survivors and nonsurvivors. The 5,10‐methylenetetrahydrofolate reductase 677C→T polymorphism had no influence on tHcy levels and survival of intensive care unit patients. Conclusions: The prevalence of hyperhomocysteinemia is increased in critically ill patients compared to age‐ and gender‐matched healthy individuals. The clinical significance of this finding remains to be determined.

G-protein β3 subunit gene ( gnb3 ) polymorphism 825c→t in patients with hypertensive crisis

ObjectiveThe polymorphism 825C→T in exon 10 of the gene GNB3 encoding the &bgr;3 subunit of heterotrimeric guanine nucleotide binding regulatory proteins (G-proteins) results in a splicing variant (GNB3-s) in which the nucleotides 498–620 of exon 9 are deleted. The T allele has been shown to be overrepresented in patients with essential hypertension. Because GNB3-s may support the development of severe elevation of blood pressure, we hypothesized that GNB3 825C→T may be present more frequently in patients with hypertensive crisis. DesignCase control study. SettingDepartment of Emergency Medicine at the University Hospital of Vienna, Vienna, Austria. PatientsA total of 174 patients admitted to an emergency department for treatment of hypertensive crisis diagnosed as suffering from essential hypertension. InterventionsNone. Measurements and Main ResultsPatients were genotyped for the 825C→T transition in GNB3. An equal number of age- and gender-matched normotensive, healthy individuals served as the control population. The allele frequency of 825C→T in the GNB3 gene was 0.310 in patients with hypertensive crisis and 0.342 in the control group. There was no difference in genotype distribution and allele frequency between the patients and the age- and gender-matched control group or between the observed prevalence and the occurrence rate expected from the Hardy-Weinberg principle within each group. ConclusionsGNB3 825C→T is not associated with the phenotype of hypertensive crisis in patients suffering from essential hypertension. Furthermore, our data do not support the concept that the 825C→T transition in the GNB3 gene is associated with essential hypertension.

Molecular genetic differentiation between primary lung cancers and lung metastases of other tumors

When solitary pulmonary tumors are observed in patients with a history of cancer, differentiation between metastasis and primary lung cancer is crucial for appropriate therapy. Assuming that p53 mutations are conserved in metastases, mutation analysis of the p53 gene would be a valuable tool in differentiating metastases from primary carcinomas of the lung. In nine of 267 resected lung tumors, the origin of the lung tumor could not be defined histologically. Five patients had a history of colorectal carcinoma, one had a history of breast carcinoma, one had a history of soft-tissue carcinoma, and one had a history of head and neck carcinoma. One patient with a clear cell carcinoma of the lung had been surgically treated for both renal and thyroid cancer. Material from one patient with adenocarcinoma of the lung, histologically defined regional lymph nodes, and distant brain metastasis served as a control. We extracted deoxyribonucleic acid from the snap-frozen tissue of the unclassified lung tumors, from paraffin-embedded tissue of the previously removed primary cancers, and also from peripheral blood of the patients. Exons 2 to 11 of the p53 gene were amplified in separated polymerase chain reactions and directly sequenced. In all cases, the presence of germline mutations was excluded by analysis of peripheral blood deoxyribonucleic acid. The p53 mutation detected in the deoxyribonucleic acid of the lung tumor of the control patient proved to be conserved in the lymph nodes as well as in the brain metastasis. In two cases, the lung tumors exhibited a p53 mutation not present in the previously removed primary tumor and were therefore classified as new primary lung cancers. In five cases, the lung tumors proved to be metastases of the first tumor, exhibiting the identical p53 mutation. One of these lung tumor samples could be identified as a metastasis from the renal cancer, but the corresponding thyroid cancer material was different. For two cases, molecular analysis remained inconclusive. In one case, no p53 mutation could be found in the compared samples; in the other, no deoxyribonucleic acid could be extracted. Analysis of p53 mutations allowed exact classification in tumors for which standard methods failed to distinguish between metastasis or primary tumor. More than two thirds of lung tumors in patients with previous gastrointestinal carcinoma were revealed to be metastases, but second primary lung cancer could also be diagnosed. This diagnosis allowed correct surgical and adjuvant treatment of these patients.

Identification of a variable number tandem repeat region in the human T cell receptor alpha-delta (TCRAD) locus

Abstract A number of different polymorphisms have been observed in coding as well as in non-coding regions of T cell receptor (TCR) genes. We report the identification and characterization of a highly polymorphic locus in the 3′ noncoding region of the human T cell receptor α/δ (TCRAD) on chromosome 14. In 202 unrelated individuals, ten different alleles were distinguished by polymerase chain reaction (PCR) and a heterozygosity rate of 64% was calculated. Sequence analysis revealed that this polymorphic region consists of 10 bp imperfect repeat units and represents a variable number tandem repeat region (VNTR). Stable Mendelian inheritance of this novel polymorphic marker was proven in four families. The localization of this VNTR polymorphism in the TCRAD locus should make it a useful system for linkage analysis in immunological disorders with a known role of TCRAD.