Reinald Repp

Cloning and characterization of AFX, the gene that fuses to MLL in acute leukemias with a t(X;11)(q13;q23).

We report the cloning and characterization of the entire AFX gene which fuses to MLL in acute leukemias with a t(X;11)(q13;q23). AFX consists of two exons and encodes for a protein of 501 amino acids. We found that normal B- and T-cells contain similar levels of AFX mRNA and that both the MLL/AFX as well as the AFX/MLL fusion transcripts are present in the cell line and the ANLL sample with a t(X;11)(q13;q23). The single intron of the AFX gene consists of 3706 nucleotides. It contains five simple sequence repeats with lengths of at least 12 bps, a chi-like octamer sequence (GCA/TGGA/TGG) and several immunoglobulin heptamer-like sequences (GATAGTG) that are distributed throughout the entire AFX intron sequence. In the KARPAS 45 cell line the breakpoints occur at nucleotides 2913/2914 of the AFX intron and at nucleotides 4900/4901 of the breakpoint cluster region of the MLL gene. The AFX protein belongs to the forkhead protein family. It is highly homologous to the human FKHR protein, the gene of which is disrupted by the t(2;13)(q35;q14), a chromosome rearrangement characteristic of alveolar rhabdomyosarcomas. It is noteworthy that the t(X;11)(q13;q23) in the KARPAS 45 cell line and in one acute nonlymphoblastic leukemia (ANLL) disrupts the forkhead domain of the AFX protein exactly at the same amino acids as does the t(2;13)(q35;q14) in case of the FKHR protein. In addition, the 5′-part of the AFX protein contains a conserved hexapeptide motif (QIYEWM) that is homologous to the functionally important conserved hexapeptide QIYPWM upstream of the homeobox domain in Hox proteins. This motif mediates the co-operative DNA binding of Pbx family members and Hox proteins and, therefore, plays an important role in physiologic and oncogenic processes. In acute leukemias with a t(X;11)(q13;q23), this hexapeptide motif is separated from the remaining forkhead domain within the AFX protein. The predicted amino acid sequence of AFX differs significantly from the partial AFX protein sequence published previously (Genes, Chromosomes and Cancer, 1994, 11, 79 – 84). This discrepancy can be explained by the occurrence of two sequencing errors in the earlier work at nucleotide number 783 and 844 (loss of a cytosine residue or guanosine residue, respectively) that lead to two reading frame shifts.

Suppression of hepatitis B virus enhancer 1 and 2 by hepatitis C virus core protein

BACKGROUND/AIMS Epidemiological studies have shown that coinfection or superinfection with hepatitis B virus (HBV) and C virus (HCV) frequently leads to the suppression of hepatitis B virus replication. The mechanism of this phenomenon is still unclear. Shih et al. [J Virol 1993;67:5823] reported a direct suppression of HBV replication by the core protein of HCV. The target structure of HCV core protein in this system remained unclear. METHODS As HCV core protein has been shown to influence expression from transcriptional elements, we studied whether HCV core protein altered the activity of the two HBV enhancers 1 and 2. Luciferase vectors for HBV enhancers 1 or 2 were cotransfected with expression constructs for HCV core protein in murine and human hepatocyte lines. RESULTS Full-length HCV core protein suppressed the HBV enhancer 1 up to 11-fold, the enhancer 2 3-4-fold. Suppression of HBV enhancer 1 by HCV core from genotype 1b was stronger than by HCV core of genotypes 3a or 1a. Carboxyterminally truncated core proteins had lower or no suppression activity. CONCLUSIONS These data suggest that HCV core protein may directly repress transcription of the HBV RNAs. This trans-repression may contribute to suppression of HBV replication in patients coinfected with both viruses.

Identification of a new hepatitis B virus (HBV) genotype from Brazil that expresses HBV surface antigen subtype adw4

The complete genome of a hepatitis B virus (HBV) from Brazil that expressed the subtype adw4 of HBV surface antigen (HBsAg) was cloned and sequenced. The genome, termed w4B, consists of 3215 bp. The overall genetic organization of typical hepadnaviruses with four open reading frames including the preC region was found to be conserved. When comparing the w4B sequence with 19 complete HBV genomes it was, however, found to be more divergent (15%) than any other HBV sequence thus far reported. Until now, no more than 11% divergence has been reported. Distinct from the five known HBV genotypes A to E, w4B made up a new, sixth genotype. The importance of the conserved third start codon in the HBV X gene became apparent in isolate w4B. By mutation, this ATG was out of frame, and by what appears to have been a linked mutation, a new start site two codons downstream was re-established. The significance of several other mutations is discussed.

A DNA damage repair mechanism is involved in the origin of chromosomal translocations t(4;11) in primary leukemic cells

Some chromosomal translocations involved in the origin of leukemias and lymphomas are due to malfunctions of the recombinatorial machinery of immunoglobulin and T-cell receptor-genes. This mechanism has also been proposed for translocations t(4;11)(q21;q23), which are regularly associated with acute pro-B cell leukemias in early childhood. Here, reciprocal chromosomal breakpoints in primary biopsy material of fourteen t(4;11)-leukemia patients were analysed. In all cases, duplications, deletions and inversions of less than a few hundred nucleotides indicative of malfunctioning DNA repair mechanisms were observed. We concluded that these translocation events were initiated by several DNA strand breaks on both participating chromosomes and subsequent DNA repair by ‘error-prone-repair’ mechanisms, but not by the action of recombinases of the immune system.

A multiplex-PCR to identify hepatitis B virus—genotypes A–F

Eight genotypes (A-H) of hepatitis B virus (HBV) are known with variations in nucleotide sequences greater than 8%. Several recent publications found that the clinical course and outcome of antiviral therapy depended on the genotype of the infecting HBV strain. Large epidemiological studies will require the availability of a system which is rapid, reliable and can be performed on a large number of samples. We have developed a multiplex-PCR assay which uses genotype-specific primer pairs for HBV genotypes A-F. These primer pairs specifically amplified HBV DNA of the respective genotype, either in single or in multiplex-PCR. Sensitivity of the assay was in the range of 10(4) genome equivalents.

Adrenomedullin, calcitonin gene-related peptide and their receptors: evidence for a decreased placental mRNA content in preeclampsia and HELLP syndrome

OBJECTIVE The human placenta expresses a variety of vasoactive substances and neuropeptides, which play an important role in the regulation of placental blood flow in both the maternal and foetal compartment and are therefore of critical importance for foetal growth and development. Our study was planned to examine placental mRNA amounts of vasodilatory adrenomedullin (AM), calcitonin gene-related peptide (CGRP) and their receptors (AM-R and CGRP-R) in preeclampsia and HELLP syndrome (hemolysis, elevated liver enzymes, low platelets). These are severe maternal conditions leading to an altered uteroplacental and fetoplacental perfusion and a higher risk for foetal growth retardation, premature delivery, infant mortality, and even maternal death. STUDY DESIGN We included 17 patients with preeclampsia, four women with HELLP syndrome and 34 controls. After delivery, the mRNA levels of AM, AM-R, CGRP, CGRP-R, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and -actin were measured in placental villi and chorionic plates using quantitative real-time PCR. RESULTS AM/-actin and AM/GAPDH mRNA ratios were significantly lower in placental villi in preeclampsia than in controls (P<0.05) as were CGRP/-actin and CGRP/GAPDH mRNA ratios in chorionic plates (P<0.05). Placental AM-R and CGRP-R mRNA amounts were unaffected. CONCLUSION Our data show a reduction of AM and CGRP mRNAs in contrast to unchanged mRNA levels of their receptors in placenta specimens of women with preeclampsia or HELLP syndrome.

Quantitation of gene expression by real-time PCR disproves a "retroviral hypothesis" for childhood-onset diabetes mellitus.

Children with insulin-dependent diabetes mellitus (IDDM) suffer from a chronic autoimmune β cell destruction of unknown origin, maybe due to superantigens or retroviral endogenous genes. Recently, a novel endogenous retrovirus designated as IDDMK1,222 was proposed to encode for such a candidate autoimmune gene in type 1 diabetes. We therefore analyzed the expression of IDDMK1,222 genes in peripheral blood leukocytes and plasma from 55 healthy children and 55 diabetic children including 11 patients with acute disease onset. In our study we applied an improved quantitative and highly specific real-time PCR assay. In contrast to previous data obtained by conventional PCR, IDDMK1,222 gene expression did not differ between diabetic and nondiabetic individuals. For this reason, we propose that IDDMK1,222 is an ubiquitous endogenous retroviral element in the human genome but not a candidate autoimmune gene for IDDM, especially in childhood-onset disease. Real-time PCR proved to be a highly sensitive and specific method for detection and quantitation of very low amounts of mRNA and will thereby be useful regarding the special demands in pediatric studies dealing with very low amounts of specimen.

Comparison of methylene blue, riboflavin, and N-acetylcysteine for the reduction of nitric oxide-induced methemoglobinemia

Objective: To investigate the treatment of nitric oxide (NO)‐induced methemoglobinemia by methylene blue (MB), riboflavin, and N‐acetylcysteine (NAC) in vitro. Design: Prospective, controlled in vitro study. Setting: Research laboratory in a university hospital. Participants: Five healthy volunteers. Interventions: Generation of 16% to 18% of methemoglobin in red blood cells by NO and subsequent addition of MB, riboflavin, or NAC. Simultaneous NO (32 ppm) and MB or riboflavin exposure of red blood cells. Induction of 14% to 18% of methemoglobin in red blood cells by NO, subsequent addition of MB or riboflavin, and further incubation with NO (80 ppm). Measurements and Main Results: After discontinuation of NO, mean half‐life for methemoglobin was significantly reduced by MB from 356 mins (controls) to 5 mins (10 μM) in a dosedependent manner (p < .001). NAC did not alter the half‐life for methemoglobin, and a reduction from 356 to 168 mins was seen for 120 μM riboflavin (p < .001). Methemoglobin formation after 3 hrs of NO exposure was 4.3% ± 0.7% in controls and 0.3% ± 0.1% with 10 μM MB (p < .001); 1 μM MB attenuated methemoglobin formation to 1.9% ± 0.1% (p < .01). With riboflavin (120 μM), methemoglobin was 2.2% ± 0.5% vs. 3.2% ± 0.6% in controls (p < .001). In the presence of high methemoglobin concentrations, further methemoglobin formation was inhibited by 1 and 10 μM MB (p < .001) and attenuated by 0.1 μM MB (p < .001) but not by riboflavin. Conclusions: In vitro, NO‐induced methemoglobin formation is significantly decreased by medium (1 μM) and high (10 μM) concentrations of MB and partially by high riboflavin concentrations (120 μM). NAC and low concentrations of riboflavin do not alter methemoglobin formation.

Hematologic Features and Clinical Course of an Infant With Pearson Syndrome Caused by a Novel Deletion of Mitochondrial Dna

Objective Pearson bone marrow-pancreas syndrome (PS) is a rare, usually fatal mitochondrial disorder involving the hematopoietic system in early infancy. Due to the diversity of clinical symptoms, the diagnosis can be difficult. The authors describe a boy with severe hypoplastic anemia in whom extensive clinical, biochemical, and morphologic findings led to the diagnosis of PS, and molecular analysis revealed a novel deletion of mitochondrial DNA from nucleotide position 10.371 to 14.607. Methods The patient is a 2-year-old boy who presented at age 5 months with hypoplastic macrocytic anemia. His first months of life and the family history were uneventful. Extensive pretransfusion evaluations did not reveal a metabolic, infectious, or hematologic-neoplastic etiology, and he had no evidence of exocrine pancreatic insufficiency. However, a second bone marrow aspirate at age 7 months showed a reduced cell number, vacuolated erythroblasts and myeloblasts, and ringed sideroblasts, so PS was suspected. Results Additional molecular analysis from the boys blood leukocytes revealed a deletion of mitochondrial DNA from nucleotide position 10.371 to 14.607, which was absent in his mothers blood cells, consistent with a sporadic mutation as commonly seen in PS. The muscle histology and the respiratory chain enzymes were normal. Conclusions Mitochondriopathies should be considered in children with persistent non-neuromuscular symptoms such as unexplained refractory anemia. Due to the often-fatal course of PS, the rapid detection of mitochondrial DNA deletions is imperative for diagnosis and family counseling.

Diagnostic and scientific applications of TaqMan real-time PCR in neuroblastomas

This review summarizes the present data on the diagnostic and scientific applications of TaqMan real-time PCR in neuroblastomas, a novel fluorescence-based method for quantitation of gene expression and gene copy number variations as gene amplification and locus haploinsufficiencies. Particular emphasis is spent on precision and accuracy and the comparison of TaqMan real-time PCR with more traditional methods for quantitative PCR. Provided a proper assessment for each new marker, this method has been shown to be an easy, fast and reliable alternative to the more traditional methods for the determination of MYCN amplification and neuropeptide Y gene expression. The combination with single cell picking in the neuroblastoma tissue may be a future target of the application of real-time PCR.