Reinhard Geßner

Mutations in GATA4, NKX2.5, CRELD1, and BMP4 Are Infrequently Found in Patients With Congenital Cardiac Septal Defects

Cardiac septal defects constitute the majority ofcongenitalheartdisease(CHD)inhumansandfamilialrecurrenceisreportedtoexceed5%[Burnetal.,1998].Previously, mutations in GATA4 and NKX2.5 havebeendescribedtobepathogenicforostiumsecundumatrial septal defects (ASDII) and ventricular septaldefects(VSD)[Schottetal.,1998;Gargetal.,2003].Incontrast, CRELD1 and BMP4 constitute functionalcandidatesforregulardevelopmentoftheendocardialcushionandmutationsinthesegenescause atrioven-tricular septal defects (AVSD) in animal models andhumans [Jiao et al., 2003; Robinson et al., 2003]. Wehypothesizedthatmutationsin GATA4(NM_002052),NKX2.5 (NM_004387), CRELD1 (NM_015513), andBMP4(NM_001202)canbeidentifiedinalargecohortof patients withcongenital septal defects with a focusonASDII.Weanalyzedthecodingregionofthesefourgenesin205patientswithcongenitalseptaldefectsbysinglestrandedconformationalpolymorphism(SSCP)and sequencing. The patient cohort was assembledout of 110 patients with isolated ASDII. Of these,four subjects (3.6%) mentioned a familial history andformal segregation analysis of pedigrees suggestedan autosomal dominant inheritance. However, familyrelatives were not studied systematically. To thishomogenous ASDII patient cohort we added agroupof95individualswithdifferentcongenitalseptaldefects (60 ASDII, 22 perimembranous VSD, and13 AVSD) and concomitant minor cardiac malforma-tions (Aortic coarctation ¼CoA, persistent ductusarteriosus¼PDA or partial anomalous venousreturn¼PAPVR). These patients were included as asubgroup in a candidate gene approach reportedpreviously [Ozcelik et al., 2006]. All patients wereattending the Department for Congenital Heart Dis-ease, German Heart Institute Berlin (GHIB). Patientswith syndromic appearance and/or limb malforma-tions were excluded from the genetic study andcontrolsubjectswerematchedforethnicity.Thestudyprotocol was approved by the Institutional ReviewBoard of the GHIB and Charite´.A heterozygous c.1750C>T mutation of GATA4,which predicts p.A411V, was identified in a cauca-sian patient with multiperforated ASDII and PAPVR.After exclusion in 600 control chromosomes weconsideredthevarianttobeanovelASDIIassociatedmutation representing the fifth GATA4 mutationidentified in a patient with ASDII. The carrier was a73-year-old female with ASDII and sustained atrial

Cardiac troponin C-L29Q, related to hypertrophic cardiomyopathy, hinders the transduction of the protein kinase A dependent phosphorylation signal from cardiac troponin I to C

We investigated structural and functional aspects of the first mutation in TNNC1, coding for the calcium‐binding subunit (cTnC) of cardiac troponin, which was detected in a patient with hypertrophic cardiomyopathy [ Hoffmann B, Schmidt‐Traub H, Perrot A, Osterziel KJ & Gessner R (2001) Hum Mut17, 524]. This mutation leads to a leucine–glutamine exchange at position 29 in the nonfunctional calcium‐binding site of cTnC. Interestingly, the mutation is located in a putative interaction site for the nonphosphorylated N‐terminal arm of cardiac troponin I (cTnI) [ Finley NL, Abbott MB, Abusamhadneh E, Gaponenko V, Dong W, Seabrook G, Howarth JW, Rana M, Solaro RJ, Cheung HC et al. (1999) EJB Lett453, 107–112]. According to peptide array experiments, the nonphosphorylated cTnI arm interacts with cTnC around L29. This interaction is almost abolished by L29Q, as observed upon protein kinase A‐dependent phosphorylation of cTnI at serine 22 and serine 23 in wild‐type troponin. With CD spectroscopy, minor changes are observed in the backbone of Ca2+‐free and Ca2+‐saturated cTnC upon the L29Q replacement. A small, but significant, reduction in calcium sensitivity was detected upon measuring the Ca2+‐dependent actomyosin subfragment 1 (actoS1)‐ATPase activity and the sliding velocity of thin filaments. The maximum actoS1‐ATPase activity, but not the maximum sliding velocity, was significantly enhanced. In addition, we performed our investigations at different levels of protein kinase A‐dependent phosphorylation of cTnI. The in vitro assays mainly showed that the Ca2+ sensitivity of the actoS1‐ATPase activity, and the mean sliding velocity of thin filaments, were no longer affected by protein kinase A‐dependent phosphorylation of cTnI owing to the L29Q exchange in cTnC. The findings imply a hindered transduction of the phosphorylation signal from cTnI to cTnC.

Structural model of phospholipid-reconstituted human transferrin receptor derived by electron microscopy

BACKGROUND The transferrin receptor (TfR) regulates the cellular uptake of serum iron. Although the TfR serves as a model system for endocytosis receptors, neither crystal structure analysis nor electron microscopy has yet revealed the molecular dimensions of the TfR. To derive the first molecular model, we analyzed purified, lipid-reconstituted human TfR by high-resolution electron microscopy. RESULTS A structural model of phospholipid-reconstituted TfR was derived from 72 cryo-electron microscopic images. The TfR dimer consists of a large extracellular globular domain (6.4 x 7.5 x 10.5 nm) separated from the membrane by a thin molecular stalk (2.9 nm). A comparative protein sequence analysis suggests that the stalk corresponds to amino acid residues 89-126. Under phospholipid-reconstitution conditions, the human TfR not only integrates into vesicles, but also forms rosette-like structures called proteoparticles. Scanning transmission electron microscopy revealed an overall diameter of 31.5 nm and a molecular mass of 1669 +/- 26 kDa for the proteoparticles, corresponding to nine TfR dimers. The average mass of a single receptor dimer was determined as being 186 +/- 4 kDa. CONCLUSIONS Proteoparticles resemble TfR exosomes that are expelled by sheep reticulocytes upon maturation. The structure of proteoparticles in vitro is thus interpreted as being the result of the TfRs strong self-association potential, which might facilitate the endosomal sequestration of the TfR away from other membrane proteins and its subsequent return to the cell surface within tubular structures. The stalk is assumed to facilitate the tight packing of receptor molecules in coated pits and recycling tubuli.

Prevalence of cardiac beta-myosin heavy chain gene mutations in patients with hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a frequent, autosomal-dominant cardiac disease and manifests predominantly as left ventricular hypertrophy. Mutations in the cardiac beta-myosin heavy chain gene (MYH7) are responsible for the disease in about 30% of cases where mutations were identified. We clinically evaluated a large group of 147 consecutive HCM patients from three cardiology centers in Germany, Poland, and Kyrgyzstan according to the same protocol. The DNA of the patients was systematically analyzed in the whole coding region of the MYH7 gene using PCR, single-strand conformation polymorphism analysis, and automated sequencing. Eleven different missense mutations (including seven novel ones) in 11 unrelated patients were identified, showing a mutation frequency of 7.5% in the study population. We further examined the families of five patients (three of German, one of Polish, and one of Kyrgyz origin) with 32 individuals in total. We observed a clear, age-dependent penetrance with onset of disease symptoms in the fourth decade of life. Genotype–phenotype correlations were different for each mutation, whereas the majority was associated with an intermediate/malign phenotype. In conclusion, we report a systematic molecular screening of the complete MYH7 gene in a large group of consecutive HCM patients, leading to a genetic diagnosis in 38 individuals. Information about the genotype in an individual from one family could be very useful for the clinician, especially when dealing with healthy relatives in doubt of their risk about developing HCM. The increasing application of genetic screening and the increasing knowledge about genotype–phenotype correlations will hopefully lead to an improved clinical management of HCM patients.

Cytokine/Cytokine Receptor Gene Expression in Childhood Acute Lymphoblastic Leukemia Correlation of Expression and Clinical Outcome at First Disease Recurrence

Recent studies have shown that cytokines/cytokine receptors (C/CR) affect leukemic cell growth and survival. The goal of the current study was to investigate possible correlations between gene expression patterns of C/CR in leukemic cells, clinical features, and outcome in children with acute lymphoblastic leukemia (ALL) at first disease recurrence.

Heterotypic trans-interaction of LI- and E-cadherin and their localization in plasmalemmal microdomains

Cadherins are calcium-dependent adhesion molecules important for tissue morphogenesis and integrity. LI-cadherin and E-cadherin are the two prominent cadherins in intestinal epithelial cells. Whereas LI-cadherin belongs to the subfamily of 7D (seven-domain)-cadherins defined by their seven extracellular cadherin repeats and short intracellular domain, E-cadherin is the prototype of classical cadherins with five extracellular domains and a highly conserved cytoplasmic part that interacts with catenins and thereby modulates the organization of the cytoskeleton. Here, we report a specific heterotypic trans-interaction of LI- with E-cadherin, two cadherins of distinct subfamilies. Using atomic force microscopy and laser tweezer experiments, the trans-interaction of LI- and E-cadherin was characterized on the single-molecule level and on the cellular level, respectively. This heterotypic interaction showed similar binding strength (20-52 pN at 200-4000 nm/s) and lifetime (0.8 s) as the respective homotypic interactions of LI- and E-cadherin. VE-cadherin, another classical cadherin, did not bind to LI-cadherin. In enterocytes, LI-cadherin and E-cadherin are located in different membrane regions. LI-cadherin is distributed along the basolateral membrane, whereas the majority of E-cadherin is concentrated in adherens junctions. This difference in membrane distribution was also reflected in Chinese hamster ovary cells stably expressing either LI- or E-cadherin. We found that LI-cadherin is localized almost exclusively in cholesterol-rich fractions, whereas E-cadherin is excluded from these membrane fractions. Given their different membrane localization in enterocytes, the heterotypic trans-interaction of LI- and E-cadherin might play a role during development of the intestinal epithelium when the cells do not yet have elaborate membrane specializations.

Both lysine-clusters of the NH2-terminal prion-protein fragment PrP23-110 are essential for t-PA mediated plasminogen activation.

We have recently shown that the NH(2)-terminal fragment (PrP23-110) of the human cellular prion protein (PrP(c) ) stimulates t-PA mediated plasminogen activation. PrP23-110 contains an N-terminal lysine cluster (LC1; K(23),K(24), K(27)) and a C-terminal one (LC2; K(101),K(104),K(106),K(110)). To study their biological function we have substituted all lysine residues of each cluster by alanine and generated the recombinant PrP proteins PrP23-110sLC1 and PrP23-110sLC2. The ability of the mutant proteins to stimulate plasminogen activation was assayed. We found that both lysine clusters are essential for t-PA mediated plasminogen activation. We further studied the binding of soluble PrP23-110 to immobilized t-PA or plasminogen using surface plasmon resonance. The recorded binding curves could not be modeled by classical 1:1 binding kinetics suggesting oligomerisation of PrP23-110. Further plasmon resonance studies show that indeed PrP23-110 binds to itself and that glycosaminoglycans modify this interaction. Binding of t-PA or plasminogen to PrP23-110 was no longer influenced by glycosaminoglycans when PrP23-110 was immobilized on the chip surface. Thus a possible role of heparin as a cofactor in the stimulation of plasminogen activation by t-PA could be the generation of a PrP23-110 form with both lysine clusters accessible for binding of t-PA and plasminogen.

Novel two-stage screening procedure leads to the identification of a new class of transfection enhancers.

Non‐viral gene transfer efficiency is low as compared to viral vector systems. Here we describe the discovery of new drugs that are capable of enhancing non‐viral gene transfer into mammalian cells using a novel two‐stage screening procedure.