Joachim Kreuder

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.

Stenting of the Arterial Duct and Banding of the Pulmonary Arteries

Background—Outcome of patients with hypoplastic left heart (HLH) is mainly influenced by the successful first-step palliation according to the Norwood procedure. An alternative approach is heart transplantation (HTX). The feasibility of ductal stenting in newborns with duct-dependent systemic blood flow and bilateral pulmonary artery banding has been reported. But it remains to be elucidated whether this approach allows a new strategy for patients with HLH. Methods and Results—In patients with various forms of HLH (n=11) and prostaglandin E-1 administration, ductal stenting was performed with balloon expandable Jo stents or Saxx stents. Bilateral pulmonary artery banding was surgically accomplished 1 to 3 days after the transcatheter procedure. Unrestricted blood flow through the interatrial septum was secured by balloon dilatation atrial septotomy, as required. Interventional procedures were performed with no mortality. Stent and ductal patency were achieved for up to 331 days. Two patients underwent HTX, and 8 patients had a palliative 1-stage procedure with reconstruction of the aortic arch and bidirectional cavopulmonary connection at the age of 3.5 to 6 months. There were 2 deaths. One patient with preoperative right heart failure died after the reconstructive surgery, and 1 patient died 4 months after ductal stenting and bilateral banding awaiting HTX. Conclusions—The present study is the first clinical trial showing that stenting the duct followed by bilateral pulmonary artery banding in newborns with HLH allows the combination of neoaortic reconstruction, which is part of first-stage palliation of HLH, with the establishment of a bidirectional cavopulmonary connection. Additionally, it allows the chance for HTX after extended waiting periods.

Stenting of the ductus arteriosus and banding of the pulmonary arteries: basis for various surgical strategies in newborns with multiple left heart obstructive lesions

Objective: To present an institutional experience with stent placement in the arterial duct combined with bilateral banding of the pulmonary artery branches as a basis for various surgical strategies in newborns with hypoplastic left heart obstructive lesions. Design: Observational study. Setting: Paediatric heart centre in a university hospital. Patients: 20 newborns with various forms of left heart obstructive lesions and duct dependent systemic blood flow. Interventions: Patients underwent percutaneous ductal stenting and surgical bilateral pulmonary artery banding. Atrial septotomy by balloon dilatation was performed as required, in one premature baby by the transhepatic approach. Main outcome measures: Survival; numbers of and reasons for palliative and corrective cardiac surgery. Results: One patient died immediately after percutaneous ductal stenting. One patient died in connection with the surgical approach of bilateral pulmonary banding. Stent and ductal patency were achieved for up to 331 days. Two patients underwent heart transplantation and two patients died on the waiting list. Ten patients had a palliative one stage procedure with reconstruction of the aortic arch and bidirectional cavopulmonary connection at the age of 3.5–6 months. There was one death. One patient is still awaiting this approach. Two patients received biventricular repair. In one, biventricular repair will soon be provided. Conclusions: Stenting the arterial duct combined with bilateral pulmonary artery banding in newborns with hypoplastic left heart or multiple left heart obstructive lesions allows a broad variation of surgical strategies depending on morphological findings, postnatal clinical conditions, and potential ventricular growth.

Clinical and biochemical consequences of copper-histidine therapy in Menkes disease

Menkes disease (MD) is an X-linked recessively inherited neurodegenerative disorder of copper (Cu) metabolism leading to death in early childhood. Symptoms are attributed to deficient activity of Cu-dependent enzymes. Limited experience has been reported concerning clinical and biochemical consequences of parenteral treatment with copper-(histidine)2-complex (Cu-His) in MD. Cu-His was administered in a 13-week-old boy with MD by daily intramuscular injections. After 6 weeks of therapy, Cu and caeruloplasmin in serum and Cu in CSF were normalized. The excessive dopamine level in CSF was corrected after 3 months of treatment. After 6 weeks of Cu supplementation, complete reduction of epileptic discharges, improved muscular tone and increased motor activities were observed. Developmental regression stopped and was replaced by a slight progression. Death at the age of 19 months was caused by septicaemia due to a fulminant urinary tract infection; there was no evidence of chronic Cu toxicity. These findings suggest that Cu-His supplementation may be a promising palliative treatment in MD.

Level of MYC overexpression in pediatric Burkitt's lymphoma is strongly dependent on genomic breakpoint location within the MYC locus.

Increased transcriptional activity of the MYC gene is a characteristic feature of Burkitts lymphoma. Aberrant MYC expression is caused by (1) chromosomal translocation to one of the loci carrying an immunoglobulin gene, (2) mutation within the translocated allele, (3) loss of the block to transcription elongation, or (4) promoter shift. To investigate the influence of breakpoint locations within the MYC gene on MYC transcript levels, we determined both the precise genomic MYC/IGH breakpoints and the amount of MYC mRNA in 25 samples of pediatric Burkitts lymphoma with translocation t(8;14)(q24;q32). Patients with breakpoints that were 5′ from MYC exon 1 had significantly lower expression of MYC than did patients who had a breakpoint within exon 1 or intron 1 (P < 0.05 and 0.005, respectively). The highest mRNA level of MYC (1,006 copies per 100 copies ABL1) was detected in patients with loss of the first exon and transcription initiation from a cryptic P3 promoter within the first intron of the MYC gene. In contrast, there was no obvious correlation between breakpoint locations within the IgH locus and the amount of MYC mRNA.

Assessment of pulmonary endothelial function during invasive testing in children and adolescents with idiopathic pulmonary arterial hypertension.

OBJECTIVES The purpose of our study was to assess pulmonary endothelial function by vasodilator response to acetylcholine (Ach) administered in segmental pulmonary arteries in children with idiopathic pulmonary arterial hypertension (IPAH). We hypothesized that there was a relationship among pulmonary endothelial response to Ach, severity of the disease, and clinical outcome. BACKGROUND IPAH may be associated with pulmonary endothelial dysfunction; however, data regarding the impact of endothelial dysfunction on severity and prognosis of this disease are limited. METHODS Forty-three children and adolescents (mean age: 10.4 ± 5.5 years) with IPAH were included in the study. Changes in pulmonary blood flow in response to Ach were determined using intravascular Doppler flow measurements. Pulmonary flow reserve (PFR) was calculated as the ratio of pulmonary blood flow velocity in response to Ach relative to baseline values. RESULTS Mean PFR of all patients was 1.58 ± 0.67. Mean follow-up after catheterization was 55.7 ± 41.9 months. Freedom from serious cardiovascular events (lung transplantation or death) was 83% after 2 years, 76% after 3 years, and 57% after 5 years. PFR was related significantly to World Health Organization functional class. Receiver-operating characteristic curves revealed a PFR of 1.4 as the best cutoff value. Kaplan-Meier analysis demonstrated that a PFR of <1.4 was highly predictive for cardiovascular events (log-rank [Mantel Cox] chi-square: 12.49, p < 0.0001). CONCLUSIONS Our study demonstrates a strong relationship between pulmonary endothelial response to Ach and prognosis of children with IPAH. As an adjunct to the usual testing protocol, this method provides additional information for therapeutic guidance.

Influence of stochastics on quantitative PCR in the detection of minimal residual disease

Using quantitative RT-PCR techniques, high sensitivity and precision are crucial for correct detection of low transcript levels, for example, in the evaluation of minimal residual disease (MRD). From a primary view, the greater number of PCR cycles in a nested competitive PCR assay may provide improved sensitivity of this approach when compared to real-time RT-PCR like Taqman (usually 65–75 vs 40 cycles). The very interesting results of Guo et al support this impression and clearly underscore the clinical importance of a carefully designed RTPCR method for the detection of low transcript levels. However, it remains open as to which of the two approaches may provide a superior level of validity, reliability, and freedom from bias. For instance, patient 6 from 9/12/01 was scored negative in Taqman PCR, while nested competitive PCR measured 0.1 transcripts per microgram RNA. In all, 1–5mg was subjected to cDNA synthesis in a total volume of 40ml, and 2.5ml cDNA was subsequently used in the PCR. Consequently, a single PCR tube contained 0.3125mg RNA equivalent at the most. Thus, the tube would have contained 0.03125 transcripts, a rather questionable finding. In addition, from a mathematical point of view, we would like to comment on the influence of stochastics, which significantly contributes to the accuracy of transcript quantification at low transcript levels. Since RNA molecules are distinct entities, their quantification has to consider the Poisson type of distribution, which is named after the French mathematician Siméon Denis Poisson (1781–1840) and is a borderline case of the binominal distribution for rare events. In the case of RNA quantification, the Poisson distribution predicts the frequency distribution of the number of RNA molecules that will be present for measurement in each PCR tube of a patient’s sample. In this context, stochastic fluctuations influence sensitivity as well as precision of quantification when only a small amount of quantifiable transcripts is available, for example, under the conditions of MRD. If, for example, a PCR reaction is expected to contain a single molecule of target RNA, Poisson distribution predicts probabilities of 36.8, 36.8, 18.4, 6.1, 1.5, 0.3, and 0.1% for 0, 1, 2, 3, 4, 5, and 6 molecules, respectively, to be actually present in the PCR tube. Two practical points can be derived from this theoretical background that may potentially compromise the results of Guo et al. At first, the number of measurement repetitions becomes highly important in the detection of rare transcripts and significant differences of gene transcription. Sample size can be calculated approximately from the standardized difference d1⁄4 [expected value m reference value m0]/Ovariance s, with s1⁄4 expected value in the case of Poisson distribution. Providing a type I error of a1⁄4 0.05 and a power of 80%, a sample size of at least 19, 7, and 3 repeats is required to differentiate between 2 and 1 copies, 4 and 1 copies, and 10 and 1 copies, respectively. Second, repeated measurements of a given sample with low transcript numbers should exactly reflect the Poisson type of distribution. In this work, we systematically examined the detection of distinct very low copy numbers using a plasmid vector. All experiments were performed in eight replications. The plasmid contained a 2000 bp insert that was derived from a genomic DNA rearrangement within the MLL gene. This genomic MLL rearrangement was previously cloned from an infant with translocation t(11;19). The linearized and dephosphorylated plasmid was diluted in a large volume of 1 ml in 50 mg/ml Escherichia coli t-RNA (Roche, Mannheim, Germany) to obtain concentrations ranging from 1 to 8 copies per PCR tube. Primers and probe were as follows: 50-atgaattgaacaactaggtgagcct-30 (sense), 50-attctccctgggccacctt-30 (antisense), 50FAM-tagtccgtgtctgggcaggacctgg-TAMRA-30 (probe). In all, 40 cycles of Taqman PCR were performed with standard conditions using the manufacturer’s recommendations. Primer, probes, and the plasmid standard can be obtained from AB upon request. A standard curve was separately generated using eight-fold replicates of 1:2 dilution steps with 50mg/ml E. coli t-RNA in the range between 2 and 2 copies per well. For each step between 1 and 8 copies per tube, the expected values were within the 95% confidence interval of the replicated measurements. In none of the four series, there was statistical difference between the expected values and the observed means (w-test). The index of dispersion R(t), which approximates 1 in the case of Poisson distribution, was in the acceptable range between 0.94 and 3.11 with an average value of 1.44 (Table 1). These data clearly demonstrate that, considering the properties of the Poisson distribution, Taqman real-time RT-PCR is sensitive enough to detect 1 copy per sample. In addition, its precision allows to exactly quantify even very low copy numbers when RT-PCR conditions are optimized. Our experimental data and their mathematical modelling show that the sensitivity of real-time PCR reaches the limits that are given by stochastics. The method itself can reliably measure 1 template molecule per PCR tube. Therefore, the reported enhanced sensitivity of the competitive quantitative nested RT-PCR assay by Guo et al may essentially be caused by secondary disturbances of the fluorogenic Taqman assay and not by the method of real-time RT-PCR itself. Guo et al’s suggestions to use competitive nested RT-PCR at low transcript values or especially when real-time analysis is negative seems to be a reliable approach. On the other hand, since competitive nested RT-PCR is more timeand cost-consuming than real-time RT-PCR, optimization of the realtime assay may be a worthwhile and promising approach for quantification of MRD even in clinical samples.

Kardiales Troponin I nach kardiochirurgischer Korrekturoperation im Säuglings- und Kindesalter

Background Perioperative myocardial damage is an important determinant for postoperative cardiac function and recovery. Cardiac troponin I (cTNI) is a specific marker for myocardial damage. The aim of our study was to evaluate pre- and postoperative cTNI levels, the pattern of elevation in the first four postoperative days and the prognostic value after pediatric cardiac operation. Methods Cardiac troponin I levels were measured in 115 children mean age 36±45 months (range 4 days to 189 months) undergoing elective operation of a congenital heart defect. Routine measurements were made preoperatively, immediately after cardiopulmonary bypass and serially 8, 18, 42, 90, 138 hours thereafter. Data from 13 patients undergoing surgery without cardiopulmonary bypass served as controls. Postoperative cTNI levels were correlated with intra- and postoperative parameters (such as duration of aortic crossclamping, cardiopulmonary bypass time and need for postoperative inotropic support). Results All preoperative cTNI levels were in the normal range. Postoperatively, the highest median cTNI levels were found in patients after repair of tetralogy of Fallot (TOF), atrioventricular septal defect (AVSD) and implantation of a homo- or xenograft. Postoperative cTNI levels correlated significantly with duration of cardiopulmonary bypass and aortic crossclamping, operative approach (ventriculotomy versus atriotomy) and inotropic support (p<0.0001). Peak cTNI levels were found immediately after surgery in 77.4% of our patients, 8 hours postoperative in 13.9% and at 18 hours after the surgery in 5.2% of the patients. In three children cTNI continued to increase; a secondary increase was found in one patient. Two of these children died, two had a prolonged postoperative recovery. Conclusion The postoperative level of cardiac troponin I could be used as a marker of perioperative myocardial injury caused by ischemia and operative trauma. Peak levels usually could be obtained immediately after surgery, but a further increase of cTNI during the following 18 hours may occur and is not necessarily related to impaired recovery. However still increasing cTNI levels after 18 hours postoperatively and a secondary increase as well may be used as indicators of poor outcome. Hintergrund und Ziel Der perioperative Myokardzellschaden ist ein wichtiger Faktor für die postoperative kardiale Funktion und Erholung. Neben bekannten biochemischen Markern für einen myokardialen Zellschaden (Myoglobin, CKMB) ist das für den Herzmuskelschaden zu 100% spezifische kardiale Troponin I (cTNI) in der Routinediagnostik bestimmbar. Ziel der Untersuchung war den prä- und postoperativen Verlauf und die Bedeutung von cTNI als Marker einer myokardialen Schädigung nach kardiochirurgischer Korrektur angeborener Herzfehler zu bestimmen. Methoden Prospektiv untersucht wurden cTNI Werte bei insgesamt 115 Kindern im Alter von 36±45 Monaten (4Tage bis 189 Monate), die an einem angeborenen Herzfehler operiert wurden. Ermittelt wurden die cTNI-Werte direkt präoperativ sowie 8, 18, 42, 90 und 138 Stunden nach kardiopulmonalem Bypass. Eine Kontrollgruppe bestand aus 13 ohne Herz-Lungen-Maschine operierten Patienten. Die postoperativen cTNI-Werte wurden mit intra- und postoperativen Parametern (Dauer der kardiopulmonalen Bypass- und Aortenabklemmzeit sowie dem postoperativen Katecholaminbedarf) korreliert. Ergebnisse Bei allen Patienten befanden sich die präoperativen cTNI-Werte im Normbereich. Patienten nach Korrektur einer Fallot‘schen Tetralogie, eines atrioventrikulären Septumdefekts und nach Homo- bzw. Xenograftimplantation wiesen im Median die höchsten postoperativen cTNI Werte auf. Die Höhe des postoperativen cTNI-Wertes korrelierte signifikant mit der Dauer der kardialen Bypass- und Aortenabklemmzeit, dem operativen Zugang (Ventrikulotomie versus Atriotomie) und dem postoperativen Bedarf an Katecholaminen (p<0,0001). In 77,4% der Fälle wurden die im Verlauf höchsten cTNI-Werte direkt nach der Operation gemessen, in 13,9% zum Zeitpunkt 8 Stunden und in 5,2% zum Zeitpunkt 18 Stunden nach Beendigung der Operation. Drei Patienten wiesen auch im weiteren Verlauf einen weiteren Anstieg auf, ein Patient einen sekundären Anstieg nach initialem Abfall. Zwei dieser Patienten verstarben, die anderen beiden hatten einen protrahierten stationären Verlauf. Schlussfolgerung Nach kardiochirurgischer Korrekturoperation im Kindesalter korrelierte die Höhe der postoperativ ermittelten cTNI-Werte mit der Dauer der Ischämie, der Art der Operation und dem Katecholaminbedarf und ermöglichte eine Abschätzung des perioperativen myokardialen Zellschadens. In der Mehrzahl der Patienten war der erste postoperative cTNI-Wert der im Verlauf höchste, ein weiterer Anstieg, bei klinisch unauffälligem Verlauf, ist bis 18 Stunden nach Operation möglich. In unserem Kollektiv erwies sich ein weiterer cTNI-Anstieg nach diesem Zeitpunkt oder ein sekundärer Anstieg als prognostisch ungünstig.

Rapid detection of mitochondrial deletions by long-distance polymerase chain reaction

Sir: Deletions within the mitochondrial genome may be associated with diverse clinical phenotypes ranging from maternally transmitted adult-onset diabetes mellitus and benign nonprogressive ptosis to devastating multisystem disorders such as Leigh disease, Kearns-Sayre syndrome and Pearson bone marrow/pancreas syndrome [4]. Mitochondrial DNA (mtDNA) deletions have usually been identified by Southern blot hybridisation or by amplification of multiple overlapping DNA fragments in different polymerase chain reaction (PCR) assays. In order to cover almost the entire mitochondrial genome of 16569 basepairs by a single PCR reaction we have applied the newly developed long-distance PCR protocol for amplification of mtDNA [2]. This assay mainly differs from conventional PCR by the use of the thermostable Thermus thermophilus DNA polymerase with intrinsic proofreading activity providing amplification of DNA fragments up to 40 kb. Proofreading activity corrects nucleotide misincorporation which might otherwise prematurely terminate DNA synthesis thus overcoming the limitation of DNA fragment synthesis >4-5 kh in conventional PCR reactions. In order to amplify a DNA fragment of 15662 basepairs covering all clinically relevant deletions of mtDNA reported to date, total DNA from peripheral blood mononuclear cells from healthy donors and from a patient with Pearson syndrome was used [3]. This patient suffered from refractory sideroblastic anaemia, thrombocytopenia, neutropenia and exocrine pancreatic dysfunction since early infancy. Despite resolution of haematopoetic abnormalities within the first 3 years of life, the patient died at the age of 4 years due to progredient neurodegeneration and respiratory insufficiency. Long-distance PCR was performed as follows: PCR primers were located at po1% Agarose Gel

Examiner effect on the objective structured clinical exam - a study at five medical schools

BackgroundThe Objective Structured Clinical Examination (OSCE) is increasingly used at medical schools to assess practical competencies. To compare the outcomes of students at different medical schools, we introduced standardized OSCE stations with identical checklists.MethodsWe investigated examiner bias at standardized OSCE stations for knee- and shoulder-joint examinations, which were implemented into the surgical OSCE at five different medical schools. The checklists for the assessment consisted of part A for knowledge and performance of the skill and part B for communication and interaction with the patient. At each medical faculty, one reference examiner also scored independently to the local examiner. The scores from both examiners were compared and analysed for inter-rater reliability and correlation with the level of clinical experience. Possible gender bias was also evaluated.ResultsIn part A of the checklist, local examiners graded students higher compared to the reference examiner; in part B of the checklist, there was no trend to the findings. The inter-rater reliability was weak, and the scoring correlated only weakly with the examiner’s level of experience. Female examiners rated generally higher, but male examiners scored significantly higher if the examinee was female.ConclusionsThese findings of examiner effects, even in standardized situations, may influence outcome even when students perform equally well. Examiners need to be made aware of these biases prior to examining.