Ceruloplasmin Reduces the Lactoferrin/Oleic Acid Antitumor Complex-Mediated Release of Heme-Containing Proteins from Blood Cells.

Our previous study showed that not only bovine lactoferrin (LF), the protein of milk and neutrophils, but also the human species forms complexes with oleic acid (OA) that inhibit tumor growth. Repeated injections of human LF in complex with OA (LF/8OA) to hepatoma-carrying mice decelerated tumor growth and increased animals' longevity. However, whether the effect of the LF/8OA complex is directed exclusively against malignant cells was not studied. Hence, its effect on normal blood cells was assayed, along with its possible modulation of ceruloplasmin (CP), the preferred partner of LF among plasma proteins. The complex LF/8OA (6 µM) caused hemolysis, unlike LF alone or BSA/8OA (250 µM). The activation of neutrophils with exocytosis of myeloperoxidase (MPO), a potent oxidant, was induced by 1 µM LF/8OA, whereas BSA/8OA had a similar effect at a concentration increased by an order. The egress of heme-containing proteins, i.e., MPO and hemoglobin, from blood cells affected by LF/8OA was followed by a pronounced oxidative/halogenating stress. CP, which is the natural inhibitor of MPO, added at a concentration of 2 mol per 1 mol of LF/8OA abrogated its cytotoxic effect. It seems likely that CP can be used effectively in regulating the LF/8OA complex's antitumor activity.

New Application of the Commercially Available Dye Celestine Blue B as a Sensitive and Selective Fluorescent "Turn-On" Probe for Endogenous Detection of HOCl and Reactive Halogenated Species.

Hypochlorous acid (HOCl) derived from hydrogen peroxide and chloride anion by myeloperoxidase (MPO) plays a significant role in physiological and pathological processes. Herein we report a phenoxazine-based fluorescent probe Celestine Blue B (CB) that is applicable for HOCl detection in living cells and for assaying the chlorinating activity of MPO. A remarkable selectivity and sensitivity (limit of detection is 32 nM), along with a rapid "turn-on" response of CB to HOCl was demonstrated. Furthermore, the probe was able to detect endogenous HOCl and reactive halogenated species by fluorescence spectroscopy, confocal microscopy, and flow cytometry techniques. Hence, CB is a promising tool for investigating the role of HOCl in health and disease and for screening the drugs capable of regulating MPO activity.

Caught red handed: modeling and confirmation of the myeloperoxidase ceruloplasmin alpha-thrombin complex.

The work is devoted to the study of the structural characteristics of the myeloperoxidase-ceruloplasmin-thrombin complex using small-angle neutron scattering methods in combination with computer modeling, as well as surface plasmon resonance and solid-phase enzyme assay. We have previously shown that the functioning of active myeloperoxidase during inflammation, despite the presence in the blood of an excess of ceruloplasmin which inhibits its activity, is possible due to the partial proteolysis of ceruloplasmin by thrombin. In this study, the myeloperoxidase-ceruloplasmin-thrombin heterohexamer was obtained in vitro. The building of a heterohexamer full-atomic model in silico, considering the glycosylation of the constituent proteins, confirmed the absence of steric barriers for the formation of protein-protein contacts. It was shown that the partial proteolysis of ceruloplasmin does not affect its ability to bind to myeloperoxidase, and a structural model of the heterohexamer was obtained using the small-angle neutron scattering method.

Lactoferrin Induces Erythropoietin Synthesis and Rescues Cognitive Functions in the Offspring of Rats Subjected to Prenatal Hypoxia.

The protective effects of recombinant human lactoferrin rhLF (branded "CAPRABEL™") on the cognitive functions of rat offspring subjected to prenatal hypoxia (7% O2, 3 h, 14th day of gestation) have been analyzed. About 90% of rhLF in CAPRABEL was iron-free (apo-LF). Rat dams received several injections of 10 mg of CAPRABEL during either gestation (before and after the hypoxic attack) or lactation. Western blotting revealed the appearance of erythropoietin (EPO) alongside the hypoxia-inducible factors (HIFs) in organ homogenates of apo-rhLF-treated pregnant females, their embryos (but not placentas), and in suckling pups from the dams treated with apo-rhLF during lactation. Apo-rhLF injected to rat dams either during pregnancy or nurturing the pups was able to rescue cognitive deficits caused by prenatal hypoxia and improve various types of memory both in young and adult offspring when tested in the radial maze and by the Novel Object Recognition (NOR) test. The data obtained suggested that the apo-form of human LF injected to female rats during gestation or lactation protects the cognitive functions of their offspring impaired by prenatal hypoxia.

A serine protease secreted from Bacillus subtilis cleaves human plasma transthyretin to generate an amyloidogenic fragment.

Aggregation of human wild-type transthyretin (hTTR), a homo-tetrameric plasma protein, leads to acquired senile systemic amyloidosis (SSA), recently recognised as a major cause of cardiomyopathies in 1-3% older adults. Fragmented hTTR is the standard composition of amyloid deposits in SSA, but the protease(s) responsible for amyloidogenic fragments generation in vivo is(are) still elusive. Here, we show that subtilisin secreted from Bacillus subtilis, a gut microbiota commensal bacterium, translocates across a simulated intestinal epithelium and cleaves hTTR both in solution and human plasma, generating the amyloidogenic fragment hTTR(59-127), which is also found in SSA amyloids in vivo. To the best of our knowledge, these findings highlight a novel pathogenic mechanism for SSA whereby increased permeability of the gut mucosa, as often occurs in elderly people, allows subtilisin (and perhaps other yet unidentified bacterial proteases) to reach the bloodstream and trigger generation of hTTR fragments, acting as seeding nuclei for preferential amyloid fibrils deposition in the heart.

The effect of myeloperoxidase isoforms on biophysical properties of red blood cells.

Myeloperoxidase (MPO), an oxidant-producing enzyme, stored in azurophilic granules of neutrophils has been recently shown to influence red blood cell (RBC) deformability leading to abnormalities in blood microcirculation. Native MPO is a homodimer, consisting of two identical protomers (monomeric MPO) connected by a single disulfide bond but in inflammatory foci as a result of disulfide cleavage monomeric MPO (hemi-MPO) can also be produced. This study investigated if two MPO isoforms have distinct effects on biophysical properties of RBCs. We have found that hemi-MPO, as well as the dimeric form, bind to the glycophorins A/B and band 3 protein on RBC's plasma membrane, that lead to reduced cell resistance to osmotic and acidic hemolysis, reduction in cell elasticity, significant changes in cell volume, morphology, and the conductance of RBC plasma membrane ion channels. Furthermore, we have shown for the first time that both dimeric and hemi-MPO lead to phosphatidylserine (PS) exposure on the outer leaflet of RBC membrane. However, the effects of hemi-MPO on the structural and functional properties of RBCs were lower compared to those of dimeric MPO. These findings suggest that the ability of MPO protein to influence RBC's biophysical properties depends on its conformation (dimeric or monomeric isoform). It is intriguing to speculate that hemi-MPO appearance in blood during inflammation can serve as a regulatory mechanism addressed to reduce abnormalities on RBC response, induced by dimeric MPO.

Effect of alpha-lactalbumin and lactoferrin oleic acid complexes on chromatin structural organization.

This work focuses on the study of multimeric alpha-lactalbumin oleic acid and lactoferrin oleic acid complexes. The purpose of the research is to study possible mechanisms involved in their pro-apoptotic activities, as seen in some tumor cell cultures. Complexes featuring oleic acid (OA) with human alpha-lactalbumin (hAl) or with bovine alpha-lactalbumin (bAl), and human lactoferrin (hLf) were investigated using small-angle neutron scattering (SANS). It was shown that while alpha-lactalbumin protein complexes were formed on the surface of polydisperse OA micelles, the lactoferrin complexes comprised a monodisperse system of nanoscale particles. Both hAl and hLf complexes appeared to interact with the chromatin of isolated nuclei affecting chromatin structural organization. The possible roles of these processes in the specific anti-tumor activity of these complexes are discussed.

Looking for a partner: ceruloplasmin in protein-protein interactions.

Ceruloplasmin (CP) is a mammalian blood plasma ferroxidase. More than 95% of the copper found in plasma is carried by this protein, which is a member of the multicopper oxidase family. Proteins from this group are able to oxidize substrates through the transfer of four electrons to oxygen. The essential role of CP in iron metabolism in humans is particularly evident in the case of loss-of-function mutations in the CP gene resulting in a neurodegenerative syndrome known as aceruloplasminaemia. However, the functions of CP are not limited to the oxidation of ferrous iron to ferric iron, which allows loading of the ferric iron into transferrin and prevents the deleterious reactions of Fenton chemistry. In recent years, a number of novel CP functions have been reported, and many of these functions depend on the ability of CP to form stable complexes with a number of proteins.

Neutrophil activation in response to monomeric myeloperoxidase.

Myeloperoxidase (MPO) is an oxidant-producing enzyme that can also regulate cellular functions via its nonenzymatic effects. Mature active MPO isolated from normal human neutrophils is a 145 kDa homodimer, which consists of 2 identical protomers, connected by a single disulfide bond. By binding to CD11b/CD18 integrin, dimeric MPO induces neutrophil activation and adhesion augmenting leukocyte accumulation at sites of inflammation. This study was performed to compare the potency of dimeric and monomeric MPO to elicit selected neutrophil responses. Monomeric MPO (hemi-MPO) was obtained by treating the dimeric MPO by reductive alkylation. Analysis of the crucial signal transducer, intracellular Ca2+, showed that dimeric MPO induces Ca2+ mobilization from the intracellular calcium stores of neutrophils and influx of extracellular Ca2+ whereas the effect of monomeric MPO on Ca2+ increase in neutrophils was less. It was also shown that monomeric MPO was less efficient than dimeric MPO at inducing actin cytoskeleton reorganization, cell survival, and neutrophil degranulation. Furthermore, we have detected monomeric MPO in the blood plasma of patients with acute inflammation. Our data suggest that the decomposition of dimeric MPO into monomers can serve as a regulatory mechanism that controls MPO-dependent activation of neutrophils and reduces the proinflammatory effects of MPO.

Enzymatic and bactericidal activity of myeloperoxidase in conditions of halogenative stress.

Myeloperoxidase (MPO), found mainly in neutrophils, is released in inflammation. MPO produces reactive halogen species (RHS), which are bactericidal agents. However, RHS overproduction, i.e., halogenative stress, can also damage host biomolecules, and MPO itself may be targeted by RHS. Therefore, we examined the susceptibility of MPO to inactivation by its primary products (HOCl, HOBr, HOSCN) and secondary products such as taurine monochloramine (TauCl) and taurine monobromamine (TauBr). MPO was dose-dependently inhibited up to complete inactivity by treatment with HOCl or HOBr. TauBr diminished the activity but did not eliminate it. TauCl had no effect. MPO became inactivated when producing HOCl or HOBr but not HOSCN. Taurine protected MPO against inactivation when MPO was catalyzing oxidation of Cl- to HOCl, whereas taurine failed to prevent inactivation when MPO was working with Br-, either alone or in combination with Cl-. SCN- interfered with HOCl-mediated MPO inhibition. UV-vis spectra showed that heme degradation is involved in HOCl- and HOBr-mediated MPO inactivation. A negative linear correlation between the remaining chlorinating activity of HOCl- or HOBr-modified MPO and Escherichia coli survival upon incubation with MPO/H2O2/Cl- was found. This study elucidated the possibility of MPO downregulation by MPO-derived RHS, which could counteract halogenative stress.

N-[4-(N,N,N-trimethylammonium)benzyl]chitosan chloride: Synthesis, interaction with DNA and evaluation of transfection efficiency.

А novel cationic chitosan derivative, N-[4-(N,N,N-trimethylammonium)benzyl]chitosan chloride (TMAB-CS), with different degrees of substitution (DS) was synthesized by a chemoselective interaction of 4-formyl-N,N,N-trimethylanilinium iodide with chitosan amino groups using a reductive amination method. Several factors (pH, reactant ratio, reaction time, and chitosan structure) were studied for their effects on the DS of the resulting TMAB-CS. The obtained derivatives were characterized by 1H NMR and FTIR spectroscopy. Turbidimetric titration showed enhanced solubility over a wide pH range even for low-substituted TMAB-CS. TMAB-CS provided strong DS-dependent binding of plasmid DNA. Dynamic light scattering measurements revealed the formation of stable polyplexes with hydrodynamic diameters of 200-300nm and ζ-potential of 20-30mV. TMAB-CS with relatively low DS (25%) demonstrated more pronounced transfection efficiency (up to 2000 cell/cm2) of plasmid DNA into the HEK293 cell line promoted by free TMAB-CS. The positive effects of lower DS can be related to a better polyplex dissociation within the cell. The cytotoxicity of TMAB-CS was comparable to that of the initial chitosan at concentrations up to 300ng/µL, even at high DS.

Functional link between ferroxidase activity of ceruloplasmin and protective effect of apo-lactoferrin: studying rats kept on a silver chloride diet.

Strongly pronounced argyrosis caused by adding AgCl to the feed of laboratory rats efficiently mimics the deficiency of ceruloplasmin (CP) ferroxidase activity. Bringing the concentration of AgCl in the feedstuff of lactating rats to 250 mg % and keeping their progeny (Ag-rats) for 3 months on the same silver-containing feed provided the serum iron content 1.4 times lower than that in the control group. Besides, the ferroxidase activity of CP dropped to zero. In CP purified from sera of Ag-rats two copper ions were substituted with two silver ions. Using rat models of both post-hemorrhagic and hemolytic anemia we showed that the deficiency of CP ferroxidase activity in Ag-rats affects the iron content in serum, though does not prevent the recovery of hemoglobin level accompanied by exhaustion of iron caches in liver and spleen. When apo-lactoferrin (apo-LF) was administered to Ag-rats suffering from either post-hemorrhagic or hemolytic anemia, both hemoglobin and serum iron were restored more rapidly than in the control animals. In independent experiments Ag-rats were compared with those fed on regular diet and the former displayed a prolonged 3-day stabilization of hypoxia-inducible factors 1 and 2 alpha (HIF-1a and HIF-2a) along with an increased serum concentration of erythropoietin. Introduction to Ag-rats of active CP separately or together with apo-LF reduced that effect to 1 day only. It is concluded that saturation of apo-LF with iron, provided by active CP, can strongly affect its protective capacity.

Ceruloplasmin decreases respiratory burst reaction during pregnancy.

Testing of pregnant women reveals weakening of neutrophil-mediated effector functions, such as reactive oxygen species generation. This study provides data confirming the phenomenon, gained through application of the flow cytometry technique. Key factors influencing neutrophil functional activity in blood plasma of pregnant women have not been detected so far. At the same time, concentration of ceruloplasmin - a copper-containing glycoprotein - is known to increase in blood significantly during pregnancy. We observed the negative correlation between ceruloplasmin concentration in blood plasma of pregnant women and the intensity of respiratory burst of neutrophils. Fractionation of plasma using gel-filtration revealed that ceruloplasmin-containing fraction demonstrated suppression of the respiratory burst reaction. Partial elimination of ceruloplasmin from the blood of pregnant women, performed with the help of specific antibodies and followed by immunoprecipitation, leads to an increased respiratory burst reaction. On the contrary, addition of ceruloplasmin to blood samples of healthy donors noticeably decreases the respiratory burst reaction. The results presented prove that change in ceruloplasmin level in plasma is necessary and sufficient for modulating the ability of neutrophils to produce reactive oxygen species during pregnancy.

Binding of human myeloperoxidase to red blood cells: Molecular targets and biophysical consequences at the plasma membrane level.

Myeloperoxidase (MPO) is an oxidant-producing enzyme that can also bind to cellular surface proteins. We found that band 3 protein and glycophorins A and B were the key MPO-binding targets of human red blood cells (RBCs). The interaction of MPO with RBC proteins was mostly electrostatic in nature because it was inhibited by desialation, exogenic sialic acid, high ionic strength, and extreme pH. In addition, MPO failed to interfere with the lectin-induced agglutination of RBCs, suggesting a minor role of glycan-recognizing mechanisms in MPO binding. Multiple biophysical properties of RBCs were altered in the presence of native (i.e., not hypochlorous acid-damaged) MPO. These changes included transmembrane potential, availability of intracellular Ca(2+), and lipid organization in the plasma membrane. MPO-treated erythrocytes became larger in size, structurally more rigid, and hypersensitive to acidic and osmotic hemolysis. Furthermore, we found a significant correlation between the plasma MPO concentration and RBC rigidity index in type-2 diabetes patients with coronary heart disease. These findings suggest that MPO functions as a mediator of novel regulatory mechanism in microcirculation, indicating the influence of MPO-induced abnormalities on RBC deformability under pathological stress conditions.

Biochemical and biological activity of arginine deiminase from Streptococcus pyogenes M22.

Streptococcus pyogenes (group A Streptococcus; GAS) is an important gram-positive extracellular bacterial pathogen responsible for a number of suppurative infections. This micro-organism has developed complex virulence mechanisms to avoid the host's defenses. We have previously reported that SDSC from GAS type M22 causes endothelial-cell dysfunction, and inhibits cell adhesion, migration, metabolism, and proliferation in a dose-dependent manner, without affecting cell viability. This work aimed to isolate and characterize a component from GAS type M22 supernatant that suppresses the proliferation of endothelial cells (EA.hy926). In the process of isolating a protein possessing antiproliferative activity we identified arginine deiminase (AD). Further study showed that this enzyme is most active at pH 6.8. Calculating Km and Vmax gave the values of 0.67 mmol·L(-1) and 42 s(-1), respectively. A distinctive feature of AD purified from GAS type M22 is that its optimum activity and the maximal rate of the catalytic process is close to neutral pH by comparison with enzymes from other micro-organisms. AD from GAS type M22 suppressed the proliferative activity of endothelial cells in a dose-dependent mode. At the same time, in the presence of AD, the proportion of cells in G0/G1 phase increased. When l-Arg was added at increasing concentrations to the culture medium containing AD (3 µg·mL(-1)), the enzyme's capacity to inhibit cell proliferation became partially depressed. The proportion of cells in phases S/G2 increased concomitantly, although the cells did not fully recover their proliferation activity. This suggests that AD from GAS type M22 has potential for the suppression of excessive cell proliferation.

Interaction of macrophage migration inhibitory factor with ceruloplasmin: role of labile copper ions.

Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is a target for pharmacological treatment of sepsis and malignant tumors. Inhibition of tautomerase activity of MIF in reaction with p-hydroxyphenylpyruvate (HPP) was observed in the presence of ceruloplasmin (CP), a copper-containing plasma protein. Binding labile copper ions to CP (CP+Cu(II)) is a prerequisite for MIF inhibiting. CP+Cu(II) is shown to be an uncompetitive inhibitor of MIF (Ki ~ 37 nM), which suggests formation of a complex 'MIF-HPP-CP-Cu(II)'. Filtration of CP+Cu(II) on a column with Chelex-100, otherwise the presence of high concentrations of histidine, cysteine or methionine abrogated the inhibitory effect of CP. Adding salts of Co(II) and Ni(II) that replace copper ions in the labile sites prevented the inhibitory effect of CP+Cu(II). Limited proteolysis of CP by thrombin diminished its oxidase activity in reaction with p-phenylenediamine, but endowed it with the capacity of inhibiting MIF. Covalent modification of MIF by phenylmethylsulfonyl fluoride (PMSF) resulted in binding of MIF-PMSF to CP immobilized on CM5 chip, the dissociation constant being 4.2 µM. In D-galactosamine-sensitized mice CP+Cu(II) increased the LPS-induced lethality from 54 to 100%, while administration of antibodies against MIF prevented the lethal effect. The enhancement by CP+Cu(II) of the pro-inflammatory signal of MIF is discussed.

Thrombin inhibits the anti-myeloperoxidase and ferroxidase functions of ceruloplasmin: relevance in rheumatoid arthritis.

Human ceruloplasmin (CP) is a multifunctional copper-binding protein produced in the liver. CP oxidizes Fe(2+) to Fe(3+), decreasing the concentration of Fe(2+) available for generating harmful oxidant species. CP is also a potent inhibitor of leukocyte myeloperoxidase (MPO) (Kd=130nM), a major source of oxidants in vivo. Rheumatoid arthritis (RA) is an inflammatory autoimmune disease affecting flexible joints and characterized by activation of both inflammatory and coagulation processes. Indeed, the levels of CP, MPO, and thrombin are markedly increased in the synovial fluid of RA patients. Here we show that thrombin cleaves CP in vitro at (481)Arg-Ser(482) and (887)Lys-Val(888) bonds, generating a nicked species that retains the native-like fold and the ferroxidase activity of the intact protein, whereas the MPO inhibitory function of CP is abrogated. Analysis of the synovial fluid of 24 RA patients reveals that CP is proteolytically degraded to a variable extent, with a fragmentation pattern similar to that observed with thrombin in vitro, and that proteolysis is blocked by hirudin, a highly potent and specific thrombin inhibitor. Using independent biophysical techniques, we show that thrombin has intrinsic affinity for CP (Kd=60-270nM), independent of proteolysis, and inhibits CP ferroxidase activity (KI=220±20nM). Mapping of thrombin binding sites with specific exosite-directed ligands (i.e., hirugen, fibrinogen γ'-peptide) and thrombin analogues having the exosites variably compromised (i.e., prothrombin, prethrombin-2, ßT-thrombin) reveals that the positively charged exosite-II of thrombin binds to the negatively charged upper region of CP, while the protease active site and exosite-I remain accessible. These results suggest that thrombin can exacerbate inflammation in RA by impairing the MPO inhibitory function of CP via proteolysis and by competitively inhibiting CP ferroxidase activity. Notably, local administration of hirudin, a highly potent and specifc thrombin inhibitor, reduces the concentration of active MPO in the synovial fluid of RA patients and has a beneficial effect on the clinical symptoms of the disease.

Lactoferrin, myeloperoxidase, and ceruloplasmin: complementary gearwheels cranking physiological and pathological processes.

Copper-containing plasma protein ceruloplasmin (Cp) forms a complex with lactoferrin (Lf), an iron-binding protein, and with the heme-containing myeloperoxidase (Mpo). In case of inflammation, Lf and Mpo are secreted from neutrophil granules. Among the plasma proteins, Cp seems to be the preferential partner of Lf and Mpo. After an intraperitoneal injection of Lf to rodents, the "Cp-Lf" complex has been shown to appear in their bloodstream. Cp prevents the interaction of Lf with protoplasts of Micrococcus luteus. Upon immunoprecipitation of Cp, the blood plasma becomes depleted of Lf and in a dose-dependent manner loses the capacity to inhibit the peroxidase activity of Mpo, but not the Mpo-catalyzed oxidation of thiocyanate in the (pseudo)halogenating cycle. Antimicrobial effect against E. coli displayed by a synergistic system that includes Lf and Mpo-H2O2-chloride, but not thiocyanate, as the substrate for Mpo is abrogated when Cp is added. Hence, Cp can be regarded as an anti-inflammatory factor that restrains the halogenating cycle and redirects the synergistic system Mpo-H2O2-chloride/thiocyanate to production of hypothiocyanate, which is relatively harmless for the human organism. Structure and functions of the "2Cp-2Lf-Mpo" complex and binary complexes Cp-Lf and 2Cp-Mpo in inflammation are discussed.

Proatherogenic modification of LDL by surface-bound myeloperoxidase.

One of the factors promoting oxidative/halogenating modification of low-density lipoproteins (LDL) is myeloperoxidase (MPO). We have shown previously that MPO binds to the LDL surfaces. The LDL-MPO complex is uncoupled in the presence of peptide EQIQDDCTGDED that corresponds to a fragment of apoB-100 (445-456). In this paper we studied how this peptide, as well as inhibitors and modulators of halogenating activity of MPO such as ceruloplasmin (CP), 4-aminobenzoic acid hydrazide (ABAH) and thiocyanate (SCN(-)) affect the accumulation of cholesterol and its esters in monocytes/macrophages after incubation with LDL subjected to different kinds of MPO-dependent oxidative/halogenating modification. In the presence of H2O2 and halides MPO causes stronger proatherogenic modification of LDL than exogenous reactive halogen species (HOCl and HOBr). Both monocytes, which differentiate into macrophages, and neutrophils secrete MPO in response to the presence of damaged LDL. The peptide EQIQDDCTGDED preventing interaction between MPO and LDL reduces the uptake of modified LDL and MPO by monocytes/macrophages and thus precludes the accumulation of intracellular cholesterol. Our results indicate that binding to MPO is important for LDL to become modified and acquire proatherogenic properties. The peptide EQIQDDCTGDED, CP, ABAH, and SCN(-) can play the role of anti-atherogenic factors reducing the deleterious effect of catalytically active MPO on LDL and accumulation of cholesterol in macrophages.

Familial hypercholesterolemia mutations in Petrozavodsk: no similarity to St. Petersburg mutation spectrum.

BACKGROUND: Familial hypercholesterolemia (FH) is a human monogenic disease induced by a variety of mutations with striking genetic diversity. Despite this variability recurrent mutations occur in each population studied, which allows both elucidating prevalent mutations and developing DNA diagnostic tools for the disease. Recent research of FH in St. Petersburg, Moscow and Novosibirsk (major cities in Russia) demonstrates that each megapolis has its own FH mutation spectrum sharing only small part of mutations with other populations in Russia and Europe. In order to optimize molecular-genetic diagnostic protocols for FH in Russia we studied mutation spectrum in other regions including Petrozavodsk, a smaller town in relatively close proximity to St. Petersburg. METHODS: The principal method was automated detection of single-strand conformation polymorphism followed by direct PCR amplified DNA sequencing. RESULTS: Twelve different mutations of the low density lipoprotein (LDL) receptor gene were detected in the Petrozavodsk sample (80 patients). Out of these twelve mutations, seven have never been described before (c.192_201delinsGGACTTCA, c. 195_196insT, c. 618 T > G, c. 1340C > G, c. 1686_1693delinsT, c. 1936C > A, c. 2191delG). Other five mutations (c. 58G > A, c. 925_931del, c. 1194C > T, c. 1532 T > C, c. 1920C > T) were previously characterized elsewhere. All new mutations are considered to be a probable cause of the FH in their carriers. Direct evidence of the neutral character of c.58G > A or p. (Gly20Arg) is provided for the first time. Each pathogenic mutation was a trait of its own unique pedigree and so far has not been found in other patients. CONCLUSIONS: Strikingly, out of twelve mutations characterized in the Petrozavodsk sample only one mutation, c. 925_931del, has previously been found in patients from St. Petersburg and Finland (most closely located studied populations), suggesting some common roots in origin of these populations in the past or limited gene exchange between them nowadays. No recurrent mutations were detected.