Serum 5'nucleotidase and alkaline phosphatase--highly predictive liver function tests for the diagnosis of graft-versus-host disease in bone marrow transplant recipients.

The diagnostic efficacy of five serum liver function tests (aspartate and alanine aminotransferase, alkaline phosphatase, 5' nucleotidase, and bilirubin) was investigated in 95 bone marrow transplant recipients in whom acute graft-vs-host disease was graded by the Seattle criteria. The patient population included a control group of 22 autologous transplant recipients (group I), 33 patients with no GVHD (group II), 21 patients with grades 1 and 2 GVHD (group III), 12 patients with grade 3 GVHD (group IV), and 7 patients with grade 4 GVHD (group V). Student t test analysis of the analytes among the five groups of patients showed that 5' nucleotidase and alkaline phosphatase were the best discriminants among all the possible combinations of group pairs. Peak levels of 5' nucleotidase within each group of patients correlated well with those of alkaline phosphatase in all the allogeneic transplant groups (II-V; r = 0.59), but the correlation of these with bilirubin was less frequent. Also, 5' nucleotidase and alkaline phosphatase showed significant discrimination (P less than 0.05) even between groups I and II, suggesting that they are more sensitive than the Seattle criteria in the diagnosis of GVHD. They also showed the best overall discriminatory ability by one-factor analysis of variance (ANOVA; P = 0.0001 as compared with 0.002, 0.009, and 0.04 for aspartate aminotransferase, alanine aminotransferase, and bilirubin, respectively). Receiver-operating curves of the five analytes again revealed that 5' nucleotidase and alkaline phosphatase were by far the best discriminators among the five groups of patients. Bilirubin was relatively insensitive because it was a good discriminator only between the control group and groups IV and V. The hepatocellular enzymes, alanine and aspartate aminotransferase, correlated well (r = 0.80) but discriminated poorly among the four groups of allogeneic transplant recipients (II-V), suggesting that all four groups had some measure of hepatocellular damage that was independent of the severity of GVHD.

Diagnosis of classical steroid 21-hydroxylase deficiency using an HLA-B locus-specific DNA-probe.

The HLA-B and steroid 21-hydroxylase loci are known to be closely linked. Restriction fragment length polymorphisms seen after digestion of genomic DNA with MspI and TaqI with the HLA-B locus-specific DNA-probe, pHLA-1.1, were examined in 7 nuclear families with classical steroid 21-hydroxylase deficiency. In each family 2 polymorphic hybridizing bands (corresponding to the 2 HLA-B genes) were seen. In all families, TaqI-generated polymorphisms allowed for identification of children previously shown on clinical and biochemical criteria to be affected by 21-hydroxylase deficiency from their unaffected sibs. The results were in complete agreement with the clinical diagnoses. Among the unaffected children, carriers could be distinguished from non-carriers in all cases by TaqI polymorphisms. MspI-generated polymorphisms allowed for full identification of genotypes in 5 families. In one family, MspI-generated polymorphisms could be used to identify affected from unaffected children, but could not distinguish between carriers and non-carriers. In another family, no identification of genotypes was possible by MspI-generated polymorphisms alone. The HLA-B locus-specific DNA-probe, pHLA-1.1, can be used for diagnosis and genotyping of individuals from families with 21-hydroxylase deficiency. This technique can be used as an alternative to HLA-serotyping, or in situations where HLA-serotyping is technically difficult, for example in chorionic villus samples.

Extended major histocompatibility complex haplotypes in celiac patients in the west of Ireland.

The highest reported prevalence of celiac disease (gluten-sensitive enteropathy) is found in the West of Ireland. Recent genetic data have suggested that major histocompatibility complex-linked loci may have a dominant genetic effect for disease susceptibility in this population compared with a recessive effect in other groups. To further understand the role of the MHC in celiac disease in the West of Ireland, we analyzed markers for 22 MHC haplotypes from celiac patients and compared them with 18 nontransmitted haplotypes found in the parents of celiac children, and with reported haplotypes from other populations. An extended MHC haplotype including [HLA-B8, DR3, DQw2, Bf*S, C4A*Q0, and C4B*1] accounted for 50% of celiac haplotypes but only 27% of nontransmitted parental haplotypes. Compared with other reported haplotypes in celiacs, patients from the West of Ireland show a higher prevalence of HLA-A1 as a component of this extended haplotype, suggesting that although the core haplotype is similar between Irish patients and others, the celiac population in the West of Ireland differs at other HLA loci. We did not observe any other common haplotypes among our patients unlike the situation in other populations. These differences may underlie the possible dominant effect of HLA-linked loci and the unusually high prevalence of celiac disease in the Irish population. We also found that the serum levels of complement components C3c, C4, and factor B were significantly lower among celiac patients than nonceliacs. The lower serum level of C4 appears to be related to the presence of deletions and null alleles at the C4A and C4B loci in celiacs.

Prevalence of nonclassical congenital adrenal hyperplasia among women self-referred for electrolytic treatment of hirsutism.

Nonclassical congenital adrenal hyperplasia (NCCAH) is well recognized among women who seek medical attention for hirsutism. However, the prevalence of this disorder among women self-referred for electrolytic treatment of hirsutism is unknown. We hypothesized that the prevalence of NCCAH among women attending an electrolysis clinic might be high. By measuring the morning salivary 17-hydroxyprogesterone (17-OHP) as a screening test for NCCAH in 46 women in the follicular phase of their menstrual cycle, we identified 12 subjects with a high basal salivary 17-OHP. Eleven agreed to have a 60-minute Cosyntropin-stimulation test, as did an additional 6 of 9 women with normal basal salivary 17-OHP, but with a particularly high hirsutism score. One of the women with high basal salivary 17-OHP had a 60-minute Cosyntropin response, which was diagnostic of NCCAH. She was of the Ashkenazi Jewish decent, a group previously reported to have a high prevalence of NCCAH. A second woman with high salivary 17-OPH had a Cosyntropin-stimulation response consistent with heterozygosity for 21-hydroxylase deficiency. None of the Cosyntropin-stimulation responses in those chosen for a high hirsutism score were diagnostic. Thus, 1 of 46 (2.2%) of the women who entered our study had unrecognized NCCAH, a prevalence only about 2-fold greater than that reported in the general population. Therefore, we recommend that electrolysis clinics advise clients from ethnic groups known to have a high frequency of NCCAH of the advisability of having a formal medical evaluation for NCCAH.

Quantitative analysis in molecular diagnostics.

Quantitative analysis of DNA products derived from polymerase chain reaction (PCR)-based assays depends on the careful optimization of each of the reaction parameters to achieve highly efficient amplification of target sequences. In practice, however, measurement of the accumulated PCR product is reliable only when analyses are performed at points in the exponential phase of the PCR amplification curve and before the onset of the plateau phase. The recent development of more sensitive DNA product detection systems has permitted the analysis of PCR assays after fewer amplification cycles, where the accumulation of product approaches linearity, while at the same time maintaining superior assay specificity. These methods include the use of high performance liquid chromatography, automated fluorescence detection, electrochemiluminescence, and the ligase chain reaction. Clinical applications of these methods are numerous and include diagnostic testing as well as therapeutic monitoring for neoplastic, infectious, and inherited genetic disease.

Kallikrein inhibition and C1-esterase inhibitor levels in patients with the lupus inhibitor.

It has been suggested that kallikrein inhibition may predispose patients with the lupus inhibitor to thrombosis by interfering with the Factor XII-mediated activation of plasminogen. To further investigate this suggestion, the authors measured kallikrein inhibition in 19 patients with the lupus inhibitor. They found that kallikrein inhibition was greater than 100% of that of a normal plasma pool in all patients and greater than 125% in 11 of 19. Kallikrein inhibition was significantly correlated with C1-esterase inhibitor (C1S-INH) concentration, which they measured by rocket immunoelectrophoresis (r = +0.55, P less than 0.05). In three patients the C1S-INH was more than 30% greater than the kallikrein inhibition. Crossed immunoelectrophoresis for C1S-INH in these patients' plasma revealed an electrophoretic mobility identical with that of the normal plasma pool. The authors suggest that C1S-INH-mediated kallikrein inhibition, in conjunction with other coagulation abnormalities, predisposes patients with the lupus inhibitor to thrombosis.

Association of the prothrombin G20210A mutation with factor V Leiden in a midwestern American population.

The prothrombin G20210A mutation has been identified as a risk factor for thrombosis. We studied the relationship between prothrombin G20210A and factor V Leiden mutations in patients with thrombophilia. The first 264 patients for whom these molecular diagnostic studies were requested at our institution were included in the study. For 116 of the 264 patients, additional coagulation test results were available in the laboratory database. The prothrombin G20210A mutation was found in 16 (6.1%) of the patients and the factor V Leiden mutation in 44 (16.7%). Of the 16 patients with the prothrombin G20210A mutation, 8 also carried factor V Leiden; this association was significant. In contrast, only 2 patients of the 116 with additional coagulation testing harbored more than 1 prothrombotic risk factor. These data support the hypothesis that thrombophilia is a multigenic disorder. Among unselected samples from a Midwestern population evaluated for thrombotic risk factors, the prevalence of factor V Leiden and prothrombin G20210A mutations are similar to those found in other populations in the Western world.

Molecular and endocrine characterization of a mutation involving a recombination between the steroid 21-hydroxylase functional gene and pseudogene.

The gene encoding steroid 21-hydroxylase activity, P450c21B, is located in the major histocompatibility complex (MHC) class III region, in close proximity to a highly homologous pseudogene, P450c21A. Recombinations between P450c21B and P450c21A have been shown to result in deficiency of 21-hydroxylase activity, the usual cause of congenital adrenal hyperplasia (CAH). A mutant P450c21 gene from a patient with simple virilizing CAH was identified and shown to be consistent with a recombination between P450c21A and P450c21B. Sequence analysis of the mutant gene showed the recombination site to be located between the first exon and the second intron. The mutant gene encodes a leucine instead of the normal proline at codon 31. This mutation resides on a chromosome bearing the HLA-B44 serotype. A comparison of mutations associated with HLA-B44 and that normally found with the HLA-Bw47 serotype suggests that the HLA-B44 mutations are of more ancient origin. The patient's homologous chromosome has a deletion of P450c21B. Endocrinological testing therefore allows for testing of the mutant gene in genetic isolation. Such testing demonstrated that the patient was capable of producing aldosterone and retaining sodium in response to a low-sodium diet, indicating that the mutant gene encodes an enzyme with partial 21-hydroxylase activity.

Fetal supraventricular tachycardia treated with high-dose quinidine: toxicity associated with marked elevation of the metabolite, 3(S)-3-hydroxyquinidine.

We observed a patient at 33 weeks' gestation who was administered exceptionally large doses of quinidine to treat a fetal supraventricular tachycardia. The patient had clinical evidence of quinidine toxicity at low to mid-therapeutic levels of quinidine, but markedly elevated levels of the metabolite 3(S)-3-hydroxyquinidine (3-hydroxyquinidine). This pattern is consistent with a "fast metabolism" of quinidine. There is substantial evidence that 3-hydroxyquinidine has pharmacologic activity. We conclude that elevated levels of 3-hydroxyquinidine can be associated with a clinically significant quinidine toxicity. This can occur in the presence of a nontoxic serum quinidine level. We also measured the levels of quinidine and 3-hydroxyquinidine in the amniotic fluid and cord blood. The levels in the cord blood were 31 and 27%, respectively, of those in a simultaneously drawn maternal blood sample.

Evaluation of chimerism in DNA samples by PCR amplification of D1S80 with detection by capillary electrophoresis.

BACKGROUND: Analysis of the relative amounts of donor and recipient DNA in bone marrow after bone marrow transplantation is frequently used to determine the status of the transplant. We studied the performance of an assay to quantify chimerism based on amplification of the D1S80 variable number tandem repeat marker by PCR with detection of PCR products by capillary electrophoresis (CE). METHODS AND RESULTS: Samples from potential bone marrow donors and recipients were analyzed separately and in mixtures to simulate various degrees of chimerism from 10% to 90% and subjected to PCR/CE analysis. There was excellent agreement between the measured and known relative proportions of DNA components in chimeric samples. The lower limit of sensitivity for detection of chimerism was 1%; between-runs coefficients of variation were <5%. CONCLUSIONS: Amplification of the D1S80 minisatellite by PCR with CE detection is a reliable method for determination of the relative contribution of different DNAs in mixed samples. This method is fast, quantitative, and extremely reproducible.

Chromosomal aneuploidy in leukemic blast crisis: a potential source of error in interpretation of bone marrow engraftment analysis by VNTR amplification.

BACKGROUND: Polymerase chain reaction (PCR) amplification of polymorphic microsatellite or minisatellite DNA markers has proven to be a fast, sensitive, and specific technique in post-transplantation monitoring of bone marrow engraftment, as well as early detection of residual disease and relapse. Deletion or amplification of chromosomal segments carrying marker loci, as can occur in leukemia and other hematologic malignancies, may result in loss or increased dosage of marker alleles. Examination of these marker alleles by PCR therefore may give aberrant results, which might lead to misinterpretation of bone marrow transplantation (BMT) engraftment studies. METHODS AND RESULTS: We report a case of chronic myelogenous leukemia treated by BMT. PCR amplification of the minisatellite at the apoB locus on chromosome 2 was used to monitor the donor bone marrow engraftment. The patient experienced relapse in blast crisis with a near-haploid karyotype with loss of recipient-specific apoB allele causing an aberrant PCR result for bone marrow engraftment that mimicked full donor engraftment. CONCLUSIONS: Loss or gain of polymorphic DNA markers because of chromosomal losses or gains in some hematologic malignancies may affect the interpretation of bone marrow engraftment studies by PCR. When choosing polymorphic markers for such studies, it is important to avoid those that will be affected by expected chromosomal alteration, if possible. In addition, any abberant post-transplantation typing should prompt further investigation to rule out the possibility of chromosomal aberration. Review of all pertinent laboratory studies is important to avoid misinterpretation of results from a single test for engraftment analysis.

Analysis of CYP21 coding polymorphisms in three ethnic populations: further evidence of nonamplifying CYP21 alleles among whites.

BACKGROUND: Adrenal steroid 21-hydroxylase is essential for the synthesis of both mineralocorticoids and glucocorticoids. The gene for this enzyme, CYP21, contains several frequent coding polymorphisms. Because of its essential function in steroid synthesis, polymorphisms in this enzyme might influence a variety of disease processes. However, before disease-association studies are performed, it is important to understand the frequency of these polymorphisms among normal individuals. METHODS: Using polymerase chain reaction (PCR) with restriction enzyme digestion or size length polymorphism analysis, we measured the frequencies of the +Leu(10), Arg102Lys, and Ser268Thr polymorphisms in CYP21 in healthy whites, blacks, and Indian Americans. The subjects were all young female college students participating in a study of relative risks for cardiovascular disease in these populations. RESULTS: The frequency of each polymorphism among whites, blacks, and Indian Americans were as follows: +Leu(10), 0.55, 0.96, 0.75; Arg102, 0.63, 0.97, 0.82; and Ser268, 0.92, 0.68, 0.79, respectively. With the exception of the frequencies of the Ser268Thr polymorphism among blacks and Indian Americans, there were significantly different frequencies of each polymorphism among all groups (P<.05). Among whites, the distribution of genotypes for the +Leu(10) and Arg102Lys polymorphisms deviated significantly from expected Hardy-Weinberg values because of an excess of homozygotes. CONCLUSIONS: Among the ethnic groups, there are statistically significant differences in the frequencies of these common coding polymorphisms in CYP21 that need to be considered in disease-association studies. Deviation from Hardy-Weinberg distributions might be explained by allelic dropout during PCR, a phenomenon previously reported at this locus.

Quantification of nucleic acids.

Quantification of nucleic acids is an area of growing importance in the clinical laboratory. In addition to routine measurements of total nucleic acid concentrations, quantitative techniques are used to determine the number of microorganisms in samples, the level of expression of genes, or the presence of alterations in gene dosage. This article provides an overview of current methodologies for measurement of total and specific nucleic acid concentrations.

Genotyping of apolipoprotein e.

Apolipoprotein E (apo E) is a 299-amino acid plasma protein involved in cholesterol transport and is found in chylomicrons, very low density lipopro-tein, intermediate-density lipoprotein, and high-density lipoprotein (1,1).

An overview of genetic factors influencing plasma lipid levels and coronary artery disease risk.

BACKGROUND: Coronary artery disease (CAD) is a major cause of morbidity and mortality in most Western countries and its origin involves a significant genetic component. METHODS: Genetic and epidemiologic studies have been performed to identify factors that influence the CAD risk in the population. RESULTS: The primary loci that have been demonstrated to be associated with increased CAD risk owing to genetic mutations include the low-density lipoprotein receptor, apolipoprotein B-100, and lipoprotein(a). Additional implicated loci include lipoprotein lipase, apolipoprotein CII, cholesteryl ester transfer protein, apolipoprotein AI, and lecithin-cholesterol acyl transferase. CONCLUSIONS: Numerous mutations in known genes exert a major effect on CAD risk in some patients. However, in most patients with CAD, the genetic component is believed to be attributable to the aggregate effect of loci that, individually, exert only a minor influence on lipoprotein levels.

Conjugated hyperbilirubinemia and renal function in bone marrow transplant recipients.

Although it has long been recognized that hyperbilirubinemia is enhanced in patients with impaired renal function, a clear correlation between the levels of bilirubin and creatinine in serum has never been established. We have studied this relationship in 13 bone marrow transplant recipients who had episodes of combined conjugated hyperbilirubinemia and renal impairment. Most of the patients had graft-vs-host disease and various degrees of hepatic cholestasis as evidenced by histologic examination results and abnormal liver function tests. Serial serum specimens obtained during this period showed a good correlation between the two analytes, with a mean (+/- SD) correlation coefficient of .86 +/- .10. The slopes of the regression lines, represented as bilirubin-creatinine ratios, varied widely (0.2 to 3.6), reflecting the wide variation in the degree of hyperbilirubinemia attributable to renal impairment.