Is the poly A (T>C) mutation a causative factor for misdiagnosis in second trimester prenatal diagnosis of ß-thalassemia by fetal blood analysis on high performance liquid chromatography?

We report the problems in diagnosis faced by two families referred for prenatal diagnosis of thalassemia where cordocentesis and fetal blood analysis by high performance liquid chromatography (HPLC) had to be done. The Hb A levels of the fetal blood measured by HPLC on the VARIANT™ Hemoglobin Testing System were 1.2 and 6.7%, respectively, suggestive of a heterozygous ß-thalassemia (ß-thal) fetus in the first case and a normal fetus in the second case. In one family, one of the parents had a borderline Hb A(2) level and in the other, one parent had normal RBC indices. However, DNA sequencing, done later, showed that in the first case the fetus was a compound heterozygote for the IVS-I-5 (G>C) and the polyadenylation signal site [poly A (T>C)] mutation, while in the second case, the fetus was homozygous for the poly A mutation. This emphasizes that characterization of ß-thal mutations must be done whenever one of the parents has a borderline Hb A(2) level or normal RBC indices, and one should not rely on fetal blood analysis by HPLC for prenatal diagnosis of ß-thal so as to avoid misdiagnosis.

Quantification of mRNA in whole blood by assessing recovery of RNA and efficiency of cDNA synthesis.

BACKGROUND: Current gene expression analysis relies on the assumption that the isolated RNA represents all species of mRNA in proportions equal to those in the original materials. No system is available for absolute quantification of mRNA. METHODS: We applied whole blood to 96-well filterplates to trap leukocytes. Lysis buffer containing cocktails of specific reverse primers and known concentrations of synthetic external control RNA (RNA34) was added to filterplates, and cell lysates were transferred to oligo(dT)-immobilized microplates for hybridization. We then synthesized the cDNA in the oligo(dT)-immobilized microplates from these primer sites and used the cDNA for real-time PCR. RNA34 acted as a universal control, and gene amplification results were converted to quantities of mRNA per microliter of whole blood after the recovery of RNA34 in each sample was determined. RESULTS: Under fully optimized conditions, both added RNA34 and native mRNA species exhibited approximately 10% recovery from whole blood to real-time PCR. When whole blood was stimulated ex vivo, changes in gene expression as low as 30%-40% were detected with statistical significance, and the experimental CVs were low (10%-20%). CONCLUSION: This new system to estimate mRNA copies per microliter of whole blood may allow standardization of gene-expression-based molecular diagnostics.

Polyadenylation of ribosomal RNA by Candida albicans also involves the small subunit.

BACKGROUND: Candida albicans is a polymorphic fungus causing serious infections in immunocompromised patients. It is capable of shifting from yeast to germinating forms such as hypha and pseudohypha in response to a variety of signals, including mammalian serum. We have previously shown that some of the large 25S components of ribosomal RNA in Candida albicans get polyadenylated, and this process is transiently intensified shortly after serum exposure just prior to the appearance of germination changes. RESULTS: We now present data that this process also involves the small 18S subunit of ribosomal RNA in this organism. Unlike the large 25S subunit, polyadenylation sites near the 3' end are more variable and no polyadenylation was found at the reported maturation site of 18S. Similar to 25S, one or more polyadenylated mature sized 18S molecules get intensified transiently by serum just prior to the appearance of hypha. CONCLUSIONS: The transient increase in polyadenylation of both the large and the small subunits of ribosomal RNA just prior to the appearance of hypha, raises the possibility of a role in this process.

Two mutations in the beta-globin polyadenylylation signal reveal extended transcripts and new RNA polyadenylylation sites.

Two mutations in the beta-globin poly(A) signal were identified in Israeli patients with beta +-thalassemia by sequence analysis following PCR. One is a point mutation (AATAAA----AATAAG) and the other is a 5-base-pair deletion (AATAAA----A----). The mutant genes were used to investigate the function of the poly(A) signal in vivo and to evaluate the mechanism whereby these mutations lead to a thalassemic phenotype. Analysis of RNA derived from peripheral blood demonstrated the presence of elongated RNA species in patients carrying either mutation. Other aspects of RNA processing (initiation, splicing) were unimpaired. RNA obtained from the patients carrying the point mutation contained four discrete, extended RNA species, 1500-2900 nucleotides long, which were found to be polyadenylated. Some normal cleavage-polyadenylylation was also observed. The 5-base-pair deletion completely abolished cleavage at the normal site. This deletion mutation resulted in a phenotype of beta +-thalassemia, thus providing evidence that the extended mRNAs are translatable in vivo. Furthermore, additional transcripts, greater than 5 kilobases, presumably mRNA precursors, were found in all RNA samples, including those of nonthalassemic controls. The extended transcripts of the poly(A) mutants, together with the high molecular weight precursors, suggest that the human beta-globin gene transcription unit is significantly longer than previously recognized.

Characterization of a zinc finger gene disrupted by the t(15;17) in acute promyelocytic leukemia.

The translocation t(15;17) associated with acute promyelocytic leukemia results in the fusion of the retinoic acid receptor alpha (RARA) gene to the PML gene. Characterization of PML revealed that it is a putative zinc finger protein and potential transcription factor that is commonly expressed, with at least three major transcription products. PML breakpoints cluster in two regions on either side of an alternatively spliced exon. Although leukemic cells with translocations characteristically express only one fusion product, both PML/RARA (on the 15q+ derivative chromosome) and RARA/PML (on the 17q- derivative) are transcribed.

Molecular cloning of human platelet thromboxane A synthase.

Complementary DNA coding for thromboxane A synthase was amplified by polymerase chain reaction using primers synthesized according to the partial amino acid sequences of human platelet thromboxane A synthase (Nüsing, R., Schneider-Voss, S., and Ullrich, V. (1990) Arch. Biochem. Biophys. 280, 325-330) and cloned into pBluescript SK II(-). The primary structure of human platelet enzyme was deduced from the nucleotide sequence of the cDNA. The enzyme is composed of 533 amino acids with a molecular weight of 60,487. The primary structure of the enzyme exhibited a 34-36% homology to the amino acid sequences of cytochrome P450s classified in the P450 III gene family. The highly conserved cysteine-containing sequence involved in the heme-binding site of P450 was found near the carboxyl terminus (residues 472-492). The size of the major thromboxane A synthase mRNA from human platelets and human erythroleukemia cells was estimated to be approximately 2.2 kilobases by RNA blot analysis.

Detection of polyadenylated RNA in hepatitis B virus-infected peripheral blood mononuclear cells by polymerase chain reaction.

Polymerase chain reaction (PCR) with a reverse transcriptase step characterized a specific transcription activity in hepatitis B virus (HBV)-infected peripheral blood mononuclear cells (PBMC) in two patients (1 and 2) with chronic hepatitis positive for antibody to hepatitis B core antigen (HBc). Patient 1 was also coinfected with human hepatitis delta virus. A patient who cleared HBV replication after antiviral treatment with vidarabine served as negative control. HBV-specific RNA poly A sequences were detected by PCR in PBMC of patients 1 and 2 even without detectable HBV DNA (patient 2) as shown by dot blot and PCR assays. RNA sequences were found in both the nucleus and cytoplasm. The demonstration of HBV mRNA sequences within PBMC suggests the transcription of viral DNA, in agreement with the findings of HBV surface antigen in PBMC. The results in patient 1 demonstrated HBV mRNA sequences in leukocytes even without PCR-detectable HBV DNA sequences, likely due to ongoing hepatitis delta virus replication.

Detection of pre-S1 proteins in peripheral blood mononuclear cells from patients with HBV infection.

The presence of pre-S1 proteins in peripheral blood mononuclear cell (PBMC) samples from 115 patients with different forms of hepatitis B virus (HBV) infection was investigated by Western blot. Among 67 chronic HBsAg carriers, HBV antigens were detected in the PBMC in 80% for HBsAg, 27% for HBc/e Ag and 34% for pre-S1 proteins. The detection of pre-S1 proteins in PBMC was significantly associated with the presence of serum markers of HBV replication (HBV DNA and/or DNA polymerase). In the group of 48 consecutive patients negative for serum HBsAg, but positive for anti-HBc with or without anti-HBs, HBsAg and pre-S1 proteins could be detected in PBMC. This finding was more frequent among anti-HIV-positive patients (77 and 23% of the cases, respectively) than in the negative ones (23 and 4% of the cases, respectively). The detection of HBV DNA and polyadenylated RNA in some of the PBMC samples positive for HBV proteins suggests that these proteins may be expressed in PBMC, especially during intense HBV replication. In patients negative for serum HBsAg, PBMC may constitute a reservoir of HBV.

Molecular basis of spectrin and ankyrin deficiencies in severe hereditary spherocytosis: evidence implicating a primary defect of ankyrin.

While varying degrees of spectrin deficiency have been found in the majority of patients with hereditary spherocytosis (HS), a combined severe deficiency of both spectrin and the spectrin-binding protein, ankyrin, has been reported only in two patients with severe HS. To elucidate the molecular basis of these protein deficiencies, we have studied the synthesis, assembly, and the mRNA levels of spectrin and ankyrin in peripheral blood reticulocytes in one of the previously reported probands. Pulse-labeling studies showed that in HS reticulocytes, the synthesis of alpha-spectrin was comparable with control reticulocytes while that of beta-spectrin was increased about fourfold, presumably reflecting increased erythropoietic drive. On the HS reticulocyte membrane, the amount of newly assembled spectrin was reduced to about half of the control values, presumably reflecting a decrease in the synthesis of the spectrin binding protein, ankyrin: the ankyrin synthesis was nearly absent in the cytosol and the amounts of membrane-associated ankyrin were reduced to about half of the normal values. The changes in the amounts of spectrin and ankyrin mRNAs quantitated by slot blot and Northern blot analyses were comparable with changes in the synthesis of these proteins: The alpha spectrin mRNA was within a control range and the beta-spectrin mRNA was slightly increased, while the amounts of ankyrin mRNA were reduced to about 50% of control values. We conclude that the primary defect underlying the combined spectrin and ankyrin deficiency is a deficiency of ankyrin mRNA leading to a reduced synthesis of ankyrin which, in turn, underlies the decreased assembly of spectrin on the membrane.

Patterns of spectrin transcripts in erythroid and non-erythroid cells.

Spectrin is the major protein of the membrane erythrocyte skeleton. More recently, homologous but non-identical spectrins (fodrins) were also found in various non-erythroid tissues. Spectrin mRNA in erythroid and various non-erythroid cells was examined by direct hybridization with human alpha-spectrin, beta-spectrin (erythroid spectrins), and alpha-fodrin (non-erythroid spectrin) cDNA probes. Northern blot analysis of poly (A)+ RNA revealed a distinct pattern of expression in erythroid vs. non-erythroid cells. Erythroid cells from early erythroblasts to reticulocyte stage expressed two mRNA species of beta-spectrin, whereas they expressed only a single species of alpha-spectrin, and no alpha-fodrin mRNA. In contrast, non-erythroid cells (platelets, myeloid cells, liver, muscle, heart, cerebellum, and eye lens) expressed either no alpha-spectrin mRNA or a different molecular weight transcript(s) of this gene, and a single species of alpha-fodrin mRNA. Additionally, they also expressed from none to multiple species of beta-spectrin, and these were of different molecular size(s) from that found in erythroid cells (with the exception of platelets). Transcripts of non-erythroid spectrin, alpha-fodrin, were found as a single copy only in non-erythroid tissues. Human and murine erythroleukemia cells expressed both erythroid spectrin transcripts in addition to alpha-fodrin and raise the possibility that erythroid progenitors may have the potential to express both erythroid and non-erythroid species. These data indicated that several mRNA species of beta-spectrin could be detected in both erythroid and some non-erythroid cells. Whether multiple spectrin peptides could also be found with functional heterogeneity is unclear. However, in each case, the pattern combination observed appeared to be tissue-specific.

Circulating human blood platelets retain appreciable amounts of poly (A)+ RNA.

Platelets lack a nucleus and are usually considered to be incapable of protein synthesis due to an apparent lack of messenger RNA, precluding the construction of platelet cDNA libraries and hindering the cloning of authentic platelet cDNA's. We reasoned that vestigial amounts of messenger RNA may remain in platelets when they first separate from the megakaryocyte and circulate in the peripheral blood. We isolated poly (A)+ RNA from platelets obtained by pheresis of individuals with elevated blood platelet counts due to a myeloproliferative syndrome termed essential thrombocythemia. Northern blots using probes for platelet glycoprotein Ib indicate that the poly (A)+ RNA obtained from the platelets of these donors is, in fact, derived from platelets. Cell free translation studies using the platelet poly (A)+ RNA indicate that the material is translationally active. We conclude that, contrary to prevailing information, circulating human blood platelets retain appreciable amounts of poly (A)+ RNA and that this RNA can be harvested by the described approach. The poly (A)+ RNA provides templates for the synthesis of cDNA's that code for platelet proteins.

Serum ribonucleotide binding activity in osteoarthritis.

Previous investigations assessing ribonucleotide binding in the sera of patients with diverse forms of connective tissue disease unexpectedly identified positive responses in control osteoarthritic subjects. The current study, using a radiolabelled assay, confirms the presence of a polyadenylic acid (Poly [A] binding factor(s) in the sera of a group of 50 well-characterized patients having primary or secondary forms of this disease process as contrasted with matched control subjects. The significance of such binding activity is unknown. No significantly meaningful correlation could be established between serum Poly (A) binding levels and clinical or x-ray parameters of disease.

[Poly(A) RNA distribution and the polysome-monosome ratio in the lymphocytes of healthy persons and chronic lympholeukemia patients]. / Raspredelenie poli(A) RNK i sootnoshenie polisom i monosom v limfotsitakh zdorovykh i bol'nykh khronicheskim limfoleikozom liudei.

Intact polysomes from lymphocytes of donors and patients with chronic lymphocytic leukemia (CLL) were isolated using diethyl pyrocarbonate. It has been shown that only 3/4 of ribisomes are bound to polysomes of a comparatively small size. Poly(A) RNA of the postmitochondrial fraction is largely associated with polysomes. Cyclohexamide treatment results in recruitment of monosomes into polysomes and in a shift of ribosomes in larger polysomes. There are no differences in the polysome content and size, the effect of cyclohexamide treatment and the distribution of the polysomal and non-polysomal poly(A) RNA in normal and CLL lymphocytes. It is suggested that during the transition of resting lymphocytes into the growth state, when protein synthesis increases, the mobilization of mRNP and ribosomes into polysomes is not essential.

Stability of polyadenylated mRNA in pig lymphocytes.

The stability of cytoplasmic poly(A)-containing mRNA in unstimulated pig lymphocytes has been determined by [3H]poly(U) hybridization after actinomycin D treatment. This approach has shown that the majority of the mRNA is much more unstable than has been suggested by earlier pulse-chase experiments, and has a half-life of between 1 and 2 h.

Electron microscopic observations of heterogeneous nuclear RNA of chicken reticulocytes.

Secondary structures of annealed, snapped-back and untreated chicken reticulocyte poly(A)-containing nuclear RNA were investigated by electron microscopy. Three shapes of molecules were observed: 'coat'hanger' shaped, loop-bearing and filamentous molecules. The former two shaped molecules consist of a small and large fold-back or loop plus a short hook or tail, corresponding to the poly(A) segment at the apparent middle of the structure. The third shaped molecule, mainly seen after snap-back treatment, possesses a small fold-back structure at one end only. It is suggested that the small and large fold-backs or loops of the molecule represent the 5'- and 3'-terminal hairpin structures, respectively. Two distinct sizes of molecule, small and large, irrespective of molecule shape, were observed.

In vivo degradation of rat globin messenger RNA during maturation of reticulocytes.

The degradation of globin mRNA in rat reticulocytes maturing in the peripheral blood was investigated. Poly(A) and non poly(A) portions of mRNA molecules were determined quantitatively by hybridization with radioactive poly(U) and complementary DNA, respectively. During the degradation of mRNA in vivo, it was shown that (1) globin mRNA and the bulk of RNA decrease in parallel, (2) the average chain length of poly(A) segments in the mRNA does not change, (3) the percentage of poly(A) (-) globin mRNA in total globin mRNA does not change, and (4) fragments of large molecular weight do not accumulate. Possible mechanisms of degradation of globin mRNA in the reticulocytes are discussed on the basis of these observations.