Tous les êtres vivants présentent un arsenal de défenses contre les pathogènes, et la réponse inflammatoire constitue le processus initial de cette défense, qui s’achève par la réparation des tissus lésés. Paradoxalement, un processus inflammatoire prolongé est également associé à de nombreuses pathologies comme l’athérosclérose, l’asthme, les maladies auto-immunes mais aussi certains cancers. Le recrutement excessif de leucocytes au site de l’inflammation est un processus commun à ces pathologies. Dès lors, la compréhension et la maîtrise du phénomène complexe et finement orchestré de la migration sélective des populations leucocytaires, appelée chimiotactisme, sont des enjeux majeurs de la recherche médicale contemporaine. Les récepteurs aux peptides formylés bactériens et mitochondriaux (FPRs) forment la première famille de récepteurs chimiotactiques identifiée. Elle comprend trois membres, FPR1, 2 et 3, présentant un haut niveau de similitude et partageant certains de leurs multiples ligands. Le troisième membre de ce groupe, FPR3, reste actuellement le moins bien connu. Récemment, un agoniste de FPR3, affin et spécifique, a été identifié dans le laboratoire. Il s’agit du peptide F2L, qui correspond aux 21 premiers acides aminés de la protéine intracellulaire HEBP1. Dans le cadre de ce travail de thèse, nous nous sommes attelé à la caractérisation approfondie du récepteur FPR3 et son ligand peptidique F2L. Dans un premier temps, et à l’aide d’anticorps validés dans le cadre de ce travail, nous avons montré que le peptide F2L induit le chimiotactisme d’un ensemble de populations leucocytaires qui expriment FPR3, dont les sous-populations de macrophages des poumons, du colon et de la peau, les éosinophiles et les cellules dendritiques plasmacytoïdes. Cette distribution suggère, pour FPR3, une fonction dans la réponse inflammatoire. Nous avons pu montrer ensuite que F2L peut être généré par la protéolyse de son précurseur, HEBP1, sous l’action de la cathepsine D des macrophages. La cathepsine D est une aspartique protéase lysosomiale impliquée dans l’homéostasie cellulaire, les processus apoptotiques et inflammatoires physiologiques et pathologiques, et dans le développement tumoral. Il s’agit désormais d’identifier dans quel compartiment et sous quelles conditions F2L est produit et sécrété. Enfin, parallèlement à ces travaux, nous avons démontré que la cathepsine G, une sérine protéase contenue dans les granules azurophiles des neutrophiles, active également le récepteur FPR3. Des résultats préliminaires suggèrent un mode d’activation alternatif du récepteur, impliquant la protéolyse d’un troisième partenaire et la génération d’un agoniste actuellement non identifié. Le couple FPR3-F2L semble dès lors impliqué dans l’induction ou la résolution de la réponse inflammatoire en recrutant les éosinophiles, monocytes, macrophages et cellules dendritiques au site de la lésion.

The main goal of the thesis was to optimize the use of agonists of the FPR receptors for cancer therapy. The aim was: (1) to find the best anchor of ligands to the cancer cells and the optimal timing of the treatment, (2) to verify the universality of agonists of FPR receptors for different types of cancer, (3) to explain the synergy between ligands of FPR and ligands of TLR by flow cytometry and (4) to verify the applicability of cytotoxic test CD45+/ PI for f-MLF-DOPE ligand.

Darbo tikslas – ištirti heterociklinių junginių, gretimose padėtyse turinčių etinil– ir formilfragmentus, ciklizacijos reakcijas su įvairiais nukleofiliniais reagentais. Darbo metu buvo rastas naujas ir efektyvus pirido[4,3–d]pirimidinų sintezės būdas kurio esmė yra 4–ariletinil–5–pirimidinkarbaldehidų terminė ar mikrobangų inicijuojama reakcija su tret–butilaminu. Parodyta, kad 2–alkinilchinolin–3–karbaldehidai dalyvauja trikomponentinėse reakcijose su pirmininiais aminais ir C– bei P-pronukleofilais sudarydami 1,2–dihidrobenzo[b][1,6]naftiridinus. Pasiūlytas naujas, universalus ir efektyvus būdas benzanuliuotoms sistemoms sintetinti panaudojant metilmerkaptoacetato kalio druską metanolyje. Rasti regioselektyvūs 5,7–dihidrofuro[3,4–d]pirimidinų, 5(H)–pirano[4,3–d]pirimidinų, 1,3–dihidrofuro[3,4–b]chinolinų ir 1H–pirano[4,3–b]chinolinų sintezės būdai tandeminių 5–egzo–dig ir 6–endo–dig ciklizacijos reakcijų pagalba. Rastas efektyvus būdas 2–(2–aril(alkil)–2–oksoetil)–1H–indol–3–karbaldehidams sintetinti iš 2–alkinil–1H–indol–3–karbaldehidų ir metanolio katalizuojant sidabro druskoms. Parodyta, kad 2–alkinilchinolin–3–karbaldehidai reaguodami su dimetilfosfitu bazinėje terpėje sudaro prisijungimo produktus dimetilhidroksi– (2–pakeistus-chinolin–3–il)metilfosfonatus. Pastarieji, esant bazės pertekliaus, persigrupuoja į atitinkamus dimetil–(2–pakeistus-chinolin–3–il)metilfosfatus. Nustatyta, kad 2–(2–piridinil)etinilchinolin–3–karbaldehidas ir 6–ariletinilpirimidin–... [toliau žr. visą tekstą] / The main aims of this investigation were to investigate cyclization reactions of electron–deficient 6–alkynylpyrimidine–5–carbaldehydes and 2–alkynylquinoline–3–carbaldehydes, and electron–rich 2–alkynylindole–3–carbaldehydes and 2–alkynylthiophene–3–carbaldehydes with N–, S–. O–, C– and P–nucleophiles. It was found, that 6–arylethinylpyrimidine–5–carbaldehydes under the treatment with tert–butylamine underwent thermal or microwave–induced cyclization reaction to form pyrido[4,3–d]pyrimidines. A novel and fast synthetic method for preparation of 1,2,3–trisubstituted 1,2–dihydrobenzo[b][1,6]naphthyridines by means of a three–component reaction between 2–alkynylquinoline–3–carbaldehydes, primary amines and C– or P–pronucleophiles was developed. It was showed, that methyl mercaptoacetate was able to trigger a novel benzannulation reaction of the starting materials. Novel, concise and regioselective synthetic methods of 5,7–dihydrofuro[3,4–d]pyrimidine, 5H–pyrano[4,3–d]pyrimidine, 1,3–dihydrofuro[3,4–b]quinolines and 1H–pyrano[4,3–b]quinolines frameworks via regioselective acetalisation/cyclization reactions of 2,4–disubstituted 6–phenylethynylpyrimidine–5–carbaldehydes and 2–alkynylquinoline–3–carbaldehydes were developed. A relatively short and efficient synthesis of 2–(2–oxoethyl)–1H–indole–3–carbaldehydes via tandem 6–endo–dig cyclization from 2–alkynylindole–3–carbaldehydes was developed. It was found that 2–alkynylquinoline–3–carbaldehydes react with dimethylphosphite in... [to full text]

The main aims of this investigation were to investigate cyclization reactions of electron–deficient 6–alkynylpyrimidine–5–carbaldehydes and 2–alkynylquinoline–3–carbaldehydes, and electron–rich 2–alkynylindole–3–carbaldehydes and 2–alkynylthiophene–3–carbaldehydes with N–, S–. O–, C– and P–nucleophiles. It was found, that 6–arylethinylpyrimidine–5–carbaldehydes under the treatment with tert–butylamine underwent thermal or microwave–induced cyclization reaction to form pyrido[4,3–d]pyrimidines. A novel and fast synthetic method for preparation of 1,2,3–trisubstituted 1,2–dihydrobenzo[b][1,6]naphthyridines by means of a three–component reaction between 2–alkynylquinoline–3–carbaldehydes, primary amines and C– or P–pronucleophiles was developed. It was showed, that methyl mercaptoacetate was able to trigger a novel benzannulation reaction of the starting materials. Novel, concise and regioselective synthetic methods of 5,7–dihydrofuro[3,4–d]pyrimidine, 5H–pyrano[4,3–d]pyrimidine, 1,3–dihydrofuro[3,4–b]quinolines and 1H–pyrano[4,3–b]quinolines frameworks via regioselective acetalisation/cyclization reactions of 2,4–disubstituted 6–phenylethynylpyrimidine–5–carbaldehydes and 2–alkynylquinoline–3–carbaldehydes were developed. A relatively short and efficient synthesis of 2–(2–oxoethyl)–1H–indole–3–carbaldehydes via tandem 6–endo–dig cyclization from 2–alkynylindole–3–carbaldehydes was developed. It was found that 2–alkynylquinoline–3–carbaldehydes react with dimethylphosphite in... [to full text] / Darbo tikslas – ištirti heterociklinių junginių, gretimose padėtyse turinčių etinil– ir formilfragmentus, ciklizacijos reakcijas su įvairiais nukleofiliniais reagentais. Darbo metu buvo rastas naujas ir efektyvus pirido[4,3–d]pirimidinų sintezės būdas kurio esmė yra 4–ariletinil–5–pirimidinkarbaldehidų terminė ar mikrobangų inicijuojama reakcija su tret–butilaminu. Parodyta, kad 2–alkinilchinolin–3–karbaldehidai dalyvauja trikomponentinėse reakcijose su pirmininiais aminais ir C– bei P-pronukleofilais sudarydami 1,2–dihidrobenzo[b][1,6]naftiridinus. Pasiūlytas naujas, universalus ir efektyvus būdas benzanuliuotoms sistemoms sintetinti panaudojant metilmerkaptoacetato kalio druską metanolyje. Rasti regioselektyvūs 5,7–dihidrofuro[3,4–d]pirimidinų, 5(H)–pirano[4,3–d]pirimidinų, 1,3–dihidrofuro[3,4–b]chinolinų ir 1H–pirano[4,3–b]chinolinų sintezės būdai tandeminių 5–egzo–dig ir 6–endo–dig ciklizacijos reakcijų pagalba. Rastas efektyvus būdas 2–(2–aril(alkil)–2–oksoetil)–1H–indol–3–karbaldehidams sintetinti iš 2–alkinil–1H–indol–3–karbaldehidų ir metanolio katalizuojant sidabro druskoms. Parodyta, kad 2–alkinilchinolin–3–karbaldehidai reaguodami su dimetilfosfitu bazinėje terpėje sudaro prisijungimo produktus dimetilhidroksi– (2–pakeistus-chinolin–3–il)metilfosfonatus. Pastarieji, esant bazės pertekliaus, persigrupuoja į atitinkamus dimetil–(2–pakeistus-chinolin–3–il)metilfosfatus. Nustatyta, kad 2–(2–piridinil)etinilchinolin–3–karbaldehidas ir 6–ariletinilpirimidin–... [toliau žr. visą tekstą]

Diss. (sammanfattning) Göteborg : Univ. , 2010.

Die Expansion des renalen Tubulointerstitiums aufgrund einer Akkumulation zellulärer Bestandteile und extrazellulärer Matrix ist eine charakteristische Eigenschaft der chronischen Nierenerkrankung (CKD) und führt zu einer Progression der Erkrankung in Richtung eines terminalen Nierenversagens. Die Fibroblasten Proliferation und ihre Transformation hin zum sekretorischen Myofibroblasten-Phänotyp stellen hierbei Schlüsselereignisse dar. Signalprozesse, die zur Induktion der Myofibroblasten führen, werden aktiv beforscht um anti-fibrotische Therapieansätze zu identifizieren. Das anti-inflammatorische Protein Annexin A1 und sein Rezeptor Formyl-Peptid Rezeptor 2 (FPR2) wurden in verschiedenen Organsystemen mit der Regulation von Fibroblastenaktivität in Verbindung gebracht, jedoch wurden ihre Expression und Funktion bei renalen fibrotischen Erkrankungen bisher nicht untersucht. Ziel der aktuellen Studie war daher die Untersuchung der renalen Annexin A1- und FPR2-Expression in einem Tiermodell des chronischen Nierenversagens, sowie die Charakterisierung der funktionellen Rolle von Annexin A1 in der Regulation des Fibroblasten Phänotyps und ihrer Syntheseleistung. Dazu wurden neugeborene Sprague-Dawley Ratten in den ersten zwei Wochen ihres Lebens entweder mit Vehikel oder mit einem Angiotensin II Typ I Rezeptor Antagonisten behandelt und ohne weitere Intervention bis zu einem Alter von 11 Monaten (CKD Ratten) gehalten. Die Regulation und Lokalisation von Annexin A1 und FPR2 wurden mit Hilfe von Real-Time PCR und Immunhistochemie erfasst. Annexin A1- und FPR2-exprimierende Zellen wurden weiter durch Doppelimmunfluoreszenzfärbungen charakterisiert. Gefärbt wurde mit Antikörpern gegen endotheliale Zellen (rat endothelial cell antigen), Makrophagen (CD 68), Fibroblasten (CD73) und Myofibroblasten (alpha-smooth muscle actin (α-sma)). Zellkulturstudien wurden an immortalisierten renalen kortikalen Fibroblasten aus Wildtyp- und Annexin A1-defizienten Mäusen, sowie an etablierten humanen und murinen renalen Fibrolasten durchgeführt. Eine Überexpression von Annexin A1 wurde durch eine stabile Transfektion erreicht. Die Expression von Annexin A1, α-sma und Kollagen 1α1 wurde durch Real-Time PCR, Western Blot und Immuhistochemie erfasst. Die Sekretion des Annexin A1 Proteins wurde nach TCA-Fällung des Zellkulturüberstandes im Western Blot untersucht. Wie zu erwarten zeigten die CKD Ratten eine geringere Anzahl an Nephronen mit deutlicher glomerulären Hypertrophie. Der tubulointerstitielle Raum war durch fibrilläres Kollagen, aktivierte Fibroblasten und inflammatorische Zellen expandiert. Parallel dazu war die mRNA Expression von Annexin A1 und Transforming growth factor beta (TGF-β) signifikant erhöht. Die Annexin A1-Lokalisation mittels Doppelimmunfluorsezenz identifizierte eine große Anzahl von CD73-positiven kortikalen Fibroblasten und eine Subpopulation von Makrophagen als Annexin A1-positiv. Die Annexin A1-Menge in Myofibroblasten und renalen Endothelien war gering. FPR2 konnte in der Mehrzahl der renalen Fibroblasten, in Myofibroblasten, in einer Subpopulation von Makrophagen und in renalen Epithelzellen nachgewiesen werden. Eine Behandlung der murinen Fibroblasten mit dem pro-fibrotischen Zytokin TGF-β führte zu einem parallelen Anstieg der α-sma-, Kollagen 1α1- und Annexin A1-Biosynthese und zu einer gesteigerten Sekretion von Annexin A1. Eine Überexpression von Annexin A1 in murinen Fibroblasten reduzierte das Ausmaß der TGF-β induzierten α-sma- und Kollagen 1α1-Biosynthese. Fibroblasten aus Annexin A1-defizienten Mäusen zeigten einen starken Myofibroblasten-Phänotyp mit einer gesteigerten Expression an α-sma und Kollagen 1α1. Der Einsatz eines Peptidantagonisten des FPR2 (WRW4) resultierte in einer Stimulation der α-sma-Biosynthese, was die Vermutung nahe legte, dass Annexin A1 FPR2-vermittelt anti-fibrotische Effekte hat. Zusammenfassend zeigen diese Ergebnisse, dass renale kortikale Fibroblasten eine Hauptquelle des Annexin A1 im renalen Interstitium und einen Ansatzpunkt für Annexin A1-Signalwege in der Niere darstellen. Das Annexin A1/FPR2-System könnte daher eine wichtige Rolle in der Kontrolle des Fibroblasten Phänotyp und der Fibroblasten Aktivität spielen und daher einen neuen Ansatz für die anti-fibrotischen pharmakologischen Strategien in der Behandlung des CKD darstellen. / Expansion of the renal tubulointerstitium due to an accumulation of cellular constituents and extracellular matrix is a characteristic feature of chronic kidney disease (CKD) and leads to the progression towards renal failure. Fibroblast proliferation and transformation to the secretory myofibroblast phenotype present key events herein. The signaling process which leads to the generation of myofibroblasts is actively investigated to identify targets for antifibrotic therapeutic strategies. The antiinflammatory protein annexin A1 and its receptor formyl peptide receptor 2 (FPR2) have been implicated in the regulation of fibroblasts from various organs but the expression and function of the two products in renal fibrotic disease have not been elucidated so far. Aim of the present study was therefore to investigate the renal expression of annexin A1 and FPR2 in an animal model of chronic kidney disease and to characterize the role of annexin A1 in the regulation of fibroblast phenotype and synthetic activity. To this end, newborn Sprague-Dawley rats were treated either with vehicle or with an angiotensin II type I receptor antagonist during the first two weeks of their life and kept without further intervention until the age of 11 month (CKD rats). Regulation and localization of annexin A1 and FPR2 were studied using real-time PCR and immunohistochemistry. Annexin A1 and FPR2 expressing cells were further characterized by double labeling immunofluorescence with markers for endothelial cells (rat endothelial cell antigen), macrophages (CD68), fibroblasts (CD73), and myofibroblasts (alpha-smooth muscle actin (α-sma)). Cell culture studies were conducted in immortalized renal cortical fibroblast derived from wildtype and from annexin A1-deficient mice as well as in established cell lines of human and murine renal fibroblasts. Overexpression of annexin A1 was achieved by stable transfection. Expression of annexin A1, α-sma and collagen 1α1 was determined using real-time PCR, Western blotting and immunohistochemistry. Secretion of annexin A1 was studied using trichloroacetic acid protein precipitation of cell culture supernatants and Western blotting. As expected, CKD rats had an overall lower number of nephrons with a marked glomerular hypertrophy. The tubulointerstitial space was expanded due to an accumulation of fibrillar collagens, activated fibroblasts and inflammatory cells. In parallel, mRNA expression for Annexin A1 and transforming growth factor beta (TGF-β) was significantly increased. Double labeling immunofluorescence localization of annexin A1 demonstrated a high abundance in CD73 positive cortical interstitial fibroblasts and in a subset of CD68 immunoreactive macrophages. The abundance in myofibroblasts and renal endothelia was low. FPR2 was found in the majority of renal fibroblasts, myofibroblasts, a subset of macrophages, and in renal endothelial cells. Treatment of cultured murine fibroblasts with the profibrotic cytokine TGF-β resulted in a parallel induction of α-sma-, collagen 1α1- and annexin A1 biosynthesis. In addition, annexin A1 secretion was markedly increased. Overexpression of annexin A1 in murine fibroblasts reduced TGF β-induced α-sma- and collagen 1α1-biosynthesis. Fibroblasts derived from annexin A1-deficient mice showed a strong myofibroblast phenotype with increased expression of both, α-sma-, and collagen 1α1. Application of a peptide antagonist of FPR2 receptor (WRW4) caused a stimulation of α-sma biosynthesis thus suggesting a role of FPR2 in the antifibrotic effects of annexin A1. In conclusion, these results identify renal cortical interstitial fibroblasts as major source and as a target for annexin A1 signalling in the kidney. The annexin A1/FPR2 signalling system may therefore play an important role in the control of fibroblast phenotype and activity and may therefore provide a novel target for antifibrotic pharmacological strategies in the treatment of CKD.

The ability of human polymorphonuclear leukocytes (PMNL; neutrophils), to sense and move to sites of infection is essential for our defense against pathogens. Cell motility is critically dependent on a dynamic remodeling of morphology. The morphological polarization toward chemoattractants, such as N-formyl-Met-Leu-Phe (fMLF), is associated with temporary extension and stabilization of lamellipodia in the direction of movement. The underlying mechanisms of cell motility are, however, still not entirely elucidated. It is therefore an urgent task to extend the present experimental evidence to give solid basis for a comprehensive model. Here it is shown that nitric oxide (NO) stimulates the morphological response of neutrophils, most likely due to transient increases in [Ca2+]i, following addition of NO-donors. This will, hypothetically, activate gelsolin and other actin filament severing proteins, leading to a subsequent decrease in filamentous actin. The incapability to efficiently turnover the actin filament network then blocks all motile activity. It is also shown that N-formyl peptide receptors on polarized neutrophils accumulate non-uniformly towards regions involved in motility. It is suggested that neutrophils use the asymmetric receptor distribution for directional sensing and sustained migration. A model for lamellipodium extension, where water fluxes play a pivotal role is presented. It is suggested that water fluxes through water-selective aquaporin (AQP) channels, contribute to the propulsive force for formation of various membrane protrusions and, thus, cell motility. It is well known that small G proteins of the Rho family GTPases play important roles in the intracellular signaling underlying cell motility. In morphologically polarized neutrophils it is shown that Cdc42, Rac2 and RhoA display spatially distinct distributions, which allows for sequential chemoattractant stimulation of neutrophil motility. The specific localizations of Rac2, Cdc42 and RhoA relative to each other and filamentous actin and fMLF receptors support the hypothesized order of activation and regulation of neutrophil cell motility. In conclusion, the detailed analysis of motility-related issues presented here provide new data allowing further refinement of previous models of neutrophil motility.

<p>The chemical cues that provide an olfactory portrait of mammalian individuals are in part detected by chemosensory receptors in the vomeronasal organ (VNO). By and large, the pertinent receptor-cue combinations used for olfactory communication are unidentified. Here we identify members of the formyl peptide receptor (FPR) family of G protein coupled receptors as candidate chemosensory receptors in the VNO of mice. We demonstrate that N-formylated mitochondrially encoded peptides presented by the major histocompatibility complex (MHC) molecule H2-M3 stimulate a subset of the VNO sensory neurons (VSNs). We show that one VNO localized FPR, Fpr-rs1, is differentially activated by strain specific variants of N-formylated peptides. We show that N-formylated peptides can function as chemosignals in a strain selective pregnancy block. We propose that this link between self-recognition peptides of the immune system and chemosensory pathways provides a possible molecular means to communicate the nature of an individual's maternal lineage or strain.</p> / Dissertation

Carbamoyl phosphate synthetase (CPS) from E. coli catalyzes the formation of carbamoyl phosphate, an intermediate in the biosynthesis of pyrimidine nucleotides and arginine, from glutamine, bicarbonate and two molecules of MgATP. This reaction is catalyzed by three separate active sites that are separated in space by ~100 Å. The transfer of ammonia and carbamate through the two intramolecular tunnels was investigated by molecular dynamics simulations and experimental characterization of mutations within. The presence of an unstable reaction intermediate, carboxyphosphate, was established. A method for studying the synchronization of the two active sites on the large subunit of CPS was developed. The potential of mean force (PMF) calculations along the ammonia and carbamate transfer pathways indicate a low free-energy path for the translocation of ammonia. The highest barrier for ammonia is 7.2 kcal/mol which corresponds to a narrow turning gate surrounded by the side chains of Cys-232, Ala-251, and Ala-314 in the large subunit. A blockage in the passageway was introduced by the triple mutant C232V/A251V/A314V, which was unable to synthesize carbamoyl phosphate. The release of phosphate is necessary for the injection of carbamate into the carbamate tunnel. Two mutants, A23F and G575F, were designed to block the migration of carbamate through carbamate tunnel. The mutants retained only 1.7 percent and 3.8 percent of the catalytic activity for the synthesis of carbamoyl phosphate relative to the wild-type CPS, respectively. Formate can be utilized by CPS in the absence of bicarbonate to form formyl phosphate. This intermediate was observed by 31P, 13C, and 1H NMR. For the three NMR methods a peak corresponding to formyl phosphate was observed at 2.15 ppm (31P) , 162.4 ppm (13C), and 8.39 and 7.94 ppm (1H). The rate of formation of formyl phosphate is 0.025 ± 0.005 s-1. Formamide was not detected in the presence of an ammonia source. Fluorescence anisotropy measurements on the C551A/S171C and C551A/S717C mutants provided insight into a possible mechanism of synchronization between the two active sites on the large subunit. The biggest fluorescence anisotropy change was observed at the N-terminal domain in the presence of AMPPNP and ATP.

Glutamate formiminotransferase deficiency (OMIM 229100) is an autosomal recessive disorder marked by clinical heterogeneity. The severe phenotype, first identified in patients of Japanese descent, includes high levels of formiminoglutamate (FIGLU) in the urine in response to histidine loading, megaloblastic anemia, and mental retardation. The mild phenotype is marked by high levels of FIGLU in the urine in the absence of histidine loading, mild developmental delay and no hematological abnormalities. The gene for human glutamate formiminotransferase-cyclodeaminase consists of 15 exons and is located at 21q22.3. The protein consists of a tetramer of dimers, with dimerization essential for both formiminotransferase and cyclodeaminase activity. / Genomic DNA extracted from cell lines from three patients with suspected glutamate formiminotransferase deficiency was analyzed by PCR and sequencing of individual exons. Cell lines WG 1758 and WG 1759 are from two siblings of Germanic descent. Both siblings are heterozygous for the mutations c457 C &rarr; T and c940 G &rarr; C. The c457 C &rarr; T changes a conserved arginine to a cysteine in a loop involved in the binding of formiminotetrahydrofolate to the enzyme. The c940 G &rarr; C mutation converts an arginine to a proline in an alpha-helix essential for the dimerization of the formiminotransferase domain. Cell line WG 1795 is from a patient of Danish descent. The patient appears to be hemizygous for a c1033 insG mutation. Quantitative PCR suggests the presence of a deletion on the other chromosome, which minimally encompasses exon 9. All of the FTCD gene changes were absent in 100 control individuals (200 alleles).