Analysis of the TID-I and TID-L Splice Variants' Expression Profile under In Vitro Differentiation of Human Mesenchymal Bone Marrow Cells into Osteoblasts.

Bone formation is a complex process regulated by a variety of pathways that are not yet fully understood. One of the proteins involved in multiple osteogenic pathways is TID (DNAJA3). The aim of this work was to study the association of TID with osteogenesis. Therefore, the expression profiles of the TID splice variants (TID-L, TID-I) and their protein products were analyzed during the proliferation and differentiation of bone marrow mesenchymal stromal cells (B-MSCs) into osteoblasts. As the reference, the hFOB1.19 cell line was used. The phenotype of B-MSCs was confirmed by the presence of CD73, CD90, and CD105 surface antigens on ~97% of cells. The osteoblast phenotype was confirmed by increased alkaline phosphatase activity, calcium deposition, and expression of ALPL and SPP1. The effect of silencing the TID gene on the expression of ALPL and SPP1 was also investigated. The TID proteins and the expression of TID splice variants were detected. After differentiation, the expression of TID-L and TID-I increased 5-fold and 3.7-fold, respectively, while their silencing resulted in increased expression of SPP1. Three days after transfection, the expression of SPP1 increased 7.6-fold and 5.6-fold in B-MSCs and differentiating cells, respectively. Our preliminary study demonstrated that the expression of TID-L and TID-I changes under differentiation of B-MSCs into osteoblasts and may influence the expression of SPP1. However, for better understanding the functional association of these results with the relevant osteogenic pathways, further studies are needed.

Telomere length in elderly Caucasians weakly correlates with blood cell counts.

BACKGROUND: Age-related decrease in bone marrow erythropoietic capacity is often accompanied by the telomere length shortening in peripheral white blood cells. However, limited and conflicting data hamper the conclusive opinion regarding this relationship. Therefore, the aim of this study was to assess an association between telomere length and peripheral blood cell count parameters in the Polish elderly population. MATERIAL AND METHODS: The substudy included 1573 of 4981 subjects aged 65 years or over, participants of the population-based PolSenior study. High-molecular-weight DNA was isolated from blood mononuclear cells. Telomere length (TL) was measured by QRT-PCR as abundance of telomere template versus a single gene copy encoding acidic ribosomal phosphoprotein P0. RESULTS: Only white blood count (WBC) was significantly different in TL tertile subgroups in all subjects (P = 0.02) and in men (P = 0.01), but not in women. Merely in men significant but weak positive correlations were found between TL and WBC (r = 0.11, P < 0.05) and RBC (r = 0.08, P < 0.05). The multiple regression analysis models confirmed a weak, independent contribution of TL to both RBC and WBC. CONCLUSIONS: In the elderly, telomere shortening limits hematopoiesis capacity to a very limited extent.

Prevalence of pathogens in sympatric Ixodes ricinus and Dermacentor reticulatus ticks in Eastern Poland and their potential impact on oral-anal contacts between ticks.

INTRODUCTION AND OBJECTIVE: Little is known about interspecific contacts between ticks. Therefore, this study focused on the investigation of factors that may influence interspecific contacts between Ixodes ricinus and Dermacentor reticulatus ticks. MATERIAL AND METHODS: Ixodes ricinus males and D. reticulatus females involved in oral-anal contacts (group I) and questing specimens with no such behaviour (group II) collected in eastern Poland were examined using molecular techniques to detect Borrelia burgdorferi s.l. (Bb), Rickettsia spp. (Rs), Anaplasma phagocytophilum, Babesia microti, and Toxoplasma gondii. RESULTS: An extremely high infection rate of Bb and Rs was determined in I. ricinus males (in groups I: 100% and 46.15% and group II: 90% and 40%, respectively) and D. reticulatus females (in group I: 84.61% and 61.53% and in group II: 90% and 20%, respectively). The prevalence of other pathogens in these ticks was substantially lower. Co-infection with pathogens was detected in approximately 53% of ticks. CONCLUSIONS: The study suggests that tick-borne pathogens may have influenced the sexual behaviour of their vectors. The oral-anal contacts between I. ricinus and D. reticulatus ticks are probably stimulated by Bb and/or Rs. The presence of five pathogens and numerous co-infections in the analysed ticks indicates a risk of various human infectious diseases in the study region. Further studies are required to clarify the implications of oral-anal interspecific tick interactions.

The Identification of Potential Immunogenic Antigens in Particular Active Developmental Stages of the Rice Weevil (Sitophilus oryzae).

BACKGROUND: The rice weevil (Sitophilus oryzae) originates from subtropical and tropical areas of Asia and Africa, but it also appears on other continents, mostly as a result of trade in rice. It may occur in grain fields as well as in storage facilities, and cause allergenic reactions. The aim of this study was to identify the potential antigens in all developmental stages of S. oryzae, which may cause an allergic response in humans. METHODS: Sera of 30 patients were tested for the presence of IgE antibodies to antigens from three life stages of the rice weevil. To identify protein fractions containing potential allergens, proteins collected from larvae, pupae, and adults separated by sex of S. oryzae were fractionated by SDS-PAGE. Then, they were probed with anti-human, anti-IgE monoclonal antibodies, fractionated by SDS-PAGE and detected by Western blotting. RESULTS: In total, 26 protein fractions of males and 22 fractions of other life stages of S. oryzae (larvae, pupae, and females) positively reacted with the examined sera. CONCLUSIONS: The conducted study showed that S. oryzae may be a source of many antigens which may cause the potential allergic reactions in humans.

Analysis of miRNAs in Osteogenesis imperfecta Caused by Mutations in COL1A1 and COL1A2: Insights into Molecular Mechanisms and Potential Therapeutic Targets.

Osteogenesis imperfecta (OI) is a group of connective tissue disorders leading to abnormal bone formation, mainly due to mutations in genes encoding collagen type I (Col I). Osteogenesis is regulated by a number of molecules, including microRNAs (miRNAs), indicating their potential as targets for OI therapy. The goal of this study was to identify and analyze the expression profiles of miRNAs involved in bone extracellular matrix (ECM) regulation in patients diagnosed with OI type I caused by mutations in COL1A1 or COL1A2. Primary skin fibroblast cultures were used for DNA purification and sequence analysis, followed by analysis of miRNA expression. Sequencing analysis revealed mutations of the COL1A1 or COL1A2 genes in all OI patients, including four previously unreported. Amongst the 40 miRNAs analyzed, 9 were identified exclusively in OI cells and 26 in both OI patients and the controls. In the latter case, the expression of six miRNAs (hsa-miR-10b-5p, hsa-miR-19a-3p, hsa-miR-19b-3p, has-miR-204-5p, has-miR-216a-5p, and hsa-miR-449a) increased, while four (hsa-miR-129-5p, hsa-miR-199b-5p, hsa-miR-664a-5p, and hsa-miR-30a-5p) decreased significantly in OI cells in comparison to their expression in the control cells. The identified mutations and miRNA expression profiles shed light on the intricate processes governing bone formation and ECM regulation, paving the way for further research and potential therapeutic advancements in OI and other genetic diseases related to bone abnormality management.

Frequency of the C677T variant of the methylenetetrahydrofolate reductase (MTHFR) gene in patients with migraine with or without aura - a preliminary report.

BACKGROUND AND PURPOSE: The aim of our study was to evaluate the frequency of the C677T variant in the methylenetetrahydrofolate reductase (MTHFR) gene in patients with migraine with or without aura and to find an association between this variant and vascular lesions in magnetic resonance imaging of the head, presence of patent foramen ovale (PFO) and increased level of homocysteine. MATERIAL AND METHODS: Ninety-one patients with migraine, aged 19-57, were investigated in this study. The MTHFR C677T variant was genotyped in this group and levels of homocysteine, folic acid and vitamin B12 were measured. Transcranial Doppler sonography with test for PFO detection by injection of air contrast during the Valsalva manoeuvre was performed in each patient. RESULTS: Frequency of the C677T variant in the MTHFR gene was similar in patients and controls. Hyperhomocysteinaemia was significantly more frequent in migraine patients with the C677T variant. The prevalence of PFO was significantly higher in migraine patients with aura and the homozygous variant of the MTHFR gene. CONCLUSIONS: Frequency of the C677T variant in the MTHFR gene was similar in patients and controls. Significantly more frequent prevalence of PFO in migraine patients with aura (with homozygous recessive genotype of MTHFR probably suggests their common genetic basis. Hyperhomocysteinaemia was significantly more frequent in migraine patients with the C677T variant, which could be an additional risk factor of this disease.

Exposure of domestic dogs and cats to ticks (Acari: Ixodida) and selected tick-borne diseases in urban and recreational areas in southern Poland.

The public health problem of tick-borne diseases has attracted much attention in recent years due to an increasing incidence in humans and animals. The aim of this study was to compare the risk of exposure to ticks and tick-borne infections in dogs and cats in recreational and urbanized areas in the Lesser Poland and Silesian Provinces. For molecular testing for the presence of the selected pathogens, 207 I. ricinus females collected from 119 dogs and 50 cats, and 2 I. hexagonus females collected from 2 domestic dogs, were examined. Overall, A. phagocytophilum was found in 3.7% of the I. ricinus specimens, B. microti in 27.1%, and B. burgdorferi s.l. in 0.9%. In urban areas of both provinces, A. phagocytophilum was found in 4.8% of the I. ricinus specimens, B. microti in 41.6% and B. burgdorferi s.l. in 3.9%. Pathogens were detected B. microti in both studied I. hexagonus specimens. These findings may indicate the important role that these animals play in the circulation of these pathogens in nature.

The First Records of Canine Babesiosis in Dogs from Dermacentor reticulatus-Free Zone in Poland.

Tick-borne microorganisms belong to important etiological agents of many infectious diseases affecting humans and animals. Among them, there are haemoprotozoans of the Babesia genus, which infect erythrocytes of a host and may cause many clinical symptoms. Canine babesiosis is an emerging tick-borne disease in Southern and Central Europe. In this study, we report two cases of symptomatic canine babesiosis caused by Babesia canis in domestic dogs from the Silesian Voivodeship, Poland, as well as the presence of Dermacentor reticulatus ticks detected on one of the Babesia-infected dogs (D. reticulatus-free zone). The molecular analysis confirmed the presence of Babesia canis in the dogs' blood, and the sequencing analysis showed that the obtained sequence is 100% identical to the sequence of Babesia canis isolate 3469 (sequence ID: KX712122.1). Our findings should raise awareness of B. canis infection among dog owners and veterinarians in the region where B. canis was not previously reported in residential, non-traveling dogs, as well as ensuring that adequate diagnostic methods are available.

The New Haplotypes of Bartonella spp. and Borrelia burgdorferi Sensu Lato Identified in Lipoptena spp. (Diptera: Hippoboscidae) Collected in the Areas of North-Eastern Poland.

Deer keds are hematophagous ectoparasites (Diptera: Hippoboscidae) that mainly parasitize Cervidae. These flies are particularly important for animal health due to the occurrence of numerous pathogenic microorganisms. They may also attack humans and their bites may cause allergenic symptoms. The aim of the study was to identify the molecular characteristics of Borrelia burgdorferi sensu lato and Bartonella spp. pathogens detected in Lipoptena spp. sampled both from the hosts and from the environment. For identification of Bartonella spp and B. burgdorferi s. l., the primers specific to the rpoB and flaB gene fragments were used, respectively. The overall prevalence of B. burgdorferi s.l. DNA in Lipoptena cervi was 14.04%, including 14.8% infection in the tested group of winged specimens. The overall prevalence of Bartonella spp. was 57.02%. The presence of these bacteria was detected in 53.5% of specimens of L. cervi and 75.7% of L. fortisetosa. The phylogenetic analysis showed five new haplotypes of the rpoB gene of Bartonella sp. isolated from L. cervi/Lipoptena fortisetosa. We also identified one new haplotype of B. afzelii and three haplotypes of B. burgdorferi isolated from winged specimens of L. cervi. This is the first study to detect the genetic material of B. burgdorferi s.l. in L. cervi in Poland and the first report on the identification of these bacteria in host-seeking specimens in the environment.

The potential risk of exposure to Borrelia garinii, Anaplasma phagocytophilum and Babesia microti in the Wolinski National Park (north-western Poland).

Ixodes ricinus (Acari: Ixodida) is the main vector in Europe of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Babesia microti. Wolinski National Park (WNP) is situated by the Baltic Sea and is frequently visited by tourists. The aim of the study was to determine the potential risk of exposure to tick borne infection with B. burgdorferi s.l., A. phagocytophilum and B. microti on the areas of WNP. In total, 394 I. ricinus were tested. The pathogens in ticks were detected by PCR, nested PCR, RFLP and sequencing. Altogether, pathogens were detected in 12.69% of the studied ticks. B. burgdorferi s.l., was shown in 0.25% of the studied I. ricinus, while A. phagocytophilum and B. microti were detected in 1.01% and 10.65% of studied ticks, respectively. Co-infection by A. phagocytophilum and B. microti was shown in only one I. ricinus nymph. Analysis of B. burgdorferi s.l., genospecies showed that 0.25% of the studied ticks were infected with Borrelia garinii. The obtained results show the potentially high human risk of exposure to tick-borne infection with B. microti, and the low potential risk of infection with B. garinii and A. phagocytophilum on the studied areas of WNP.

Exploratory study of selected nucleotide variants in GRIN1, GRIN2A and GRIN2B encoding subunits of the NMDA receptor in a targeted group of schizophrenia patients with chronic cognitive impairment.

BACKGROUND: Schizophrenia is a mental disease that affects approximately 1% of the population. Despite over 100 years of research, its pathomechanism has still not been clarified. Cognitive deficits, which are one of the symptomatic dimensions of schizophrenia, usually appear a few years before the first psychotic episode. Therefore, this is why they are probably the clinical manifestation of the primary pathomechanism of schizophrenia. It is also supposed that N-methyl-D-aspartate receptor (NMDA-R) insufficiency in the prefrontal cortex is responsible for cognitive deficits in schizophrenia. The study aimed to examine whether four selected single nucleotide variants in GRIN1, GRIN2A and GRIN2B encoding NMDA-R subunits, of which two have not been tested before, are linked with the selected clinical phenotype of cognitive dysfunction in schizophrenia. METHODS: The study included the targeted group of 117 patients diagnosed with schizophrenia, all with cognitive deficits and in symptomatic remission. DNA fragments including the studied polymorphisms of the NMDA receptors subunit genes were amplified by polymerase chain reaction and subjected to sequencing. RESULTS: The study did not confirm the presence of any of the four selected single nucleotide variants in GRIN1, GRIN2A and GRIN2B subunits of NMDA-R. CONCLUSIONS: The finding indicates that selected single nucleotide variants in GRIN2A and GRIN2B encoding subunits of the NMDA receptor are not associated with the presence of cognitive deficits in schizophrenia.

Selected single-nucleotide variants in GRIN1, GRIN2A, and GRIN2B encoding subunits of the NMDA receptor are not biomarkers of schizophrenia resistant to clozapine: exploratory study.

BACKGROUND: Schizophrenia is a common mental illness whose pathogenesis is still unknown. The vulnerability and stress model in schizophrenia assume that susceptibility to the disease is mainly associated with genes. Of the five symptomatic dimensions of schizophrenia, cognitive impairment appears to be most associated with the pathogenesis of schizophrenia. The aim of the study was to explore whether selected nucleotide variants in GRIN1, GRIN2A, and GRIN2B encoding subunits of the N-methyl-D-aspartate receptor (NMDA-R) receptor occur in a selected group of patients with treatment resistant schizophrenia with cognitive impairment. METHODS: The study included 45 patients diagnosed with super refractory schizophrenia, all with cognitive deficits and chronically psychotic. DNA fragments including the studied polymorphisms of the NMDA receptors subunit genes were amplified by polymerase chain reaction and subjected to sequencing. RESULTS: The study did not confirm the presence of any of the four selected single-nucleotide variants in GRIN1, GRIN2A, and GRIN2B subunits of NMDA-R in the study group. CONCLUSION: Results of the study indicated that the selected single-nucleotide variants are not associated both with resistance to clozapine and the presence of cognitive deficits in schizophrenia. It is possible, however, that a more extensive sequencing along with analyzing the expression of these genes may reveal different single-nucleotide variants than those assumed in the study.

Two New Haplotypes of Bartonella sp. Isolated from Lipoptena fortisetosa (Diptera: Hippoboscidae) in SE Poland.

Insects of the genus Lipoptena are parasitic arthropods with a broad host range. Due to the type of parasitism (hematophagy), their potential role as vectors of pathogens, i.e., Bartonella sp., Anaplasma phagocytophilum, Rickettsia spp., and Borrelia burgdorferi is considered. As the range of their occurrence has been changing dynamically in recent years and infestations of humans have increasingly been reported, these organisms are now the subject of numerous studies. Our research aimed to present the molecular characteristics of Bartonella sp. detected in Lipoptena fortisetosa parasitizing wild cervids in south-eastern Poland. Adults of Lipoptena spp. were collected from carcasses of roe deer and red deer between spring and autumn in 2013. The PCR method was used to detect Bartonella sp. in the insects. We report two new haplotypes of the rpoB gene of Bartonella sp. isolated from L. fortisetosa feeding on wild cervids in south-eastern Poland and the presence of this invasive ectoparasitic species in the studied area since 2013. Phylogenetic analyses of newly obtained Bartonella sp. haplotypes confirmed their unique position on the constructed tree and network topology. The rpoB gene sequences found belonging to lineage B support the view that this phylogenetic lineage represents a novel Bartonella species.

The role of sheep ked (Melophagus ovinus) as potential vector of protozoa and bacterial pathogens.

The sheep ked (Melophagus ovinus) hematophagous insect may act as a potential vector of vector-borne pathogens. The aim of this study was to detect the presence of Trypanosoma spp., Bartonella spp., Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato in sheep ked collected from sheep in Poland. In total, Trypanosoma spp. was detected in 58.91% of M. ovinus, whereas Bartonella spp. and B. burgdorferi s.l. were found in 86.82% and 1.55% of the studied insects, respectively. A. phagocytophilum was not detected in the studied material. In turn, co-infection by Trypanosoma spp. and Bartonella spp. was detected in 50.39%, while co-infection with Trypanosoma spp. and Bartonella spp. and B. burgdorferi s.l. was found in 1.55% of the studied insects. The conducted study showed for the first time the presence of B. burgdorferi s. l. in M. ovinus, as well as for the first time in Poland the presence of Trypanosoma spp. and Bartonella spp. The obtained results suggest that these insects may be a potential vector for these pathogens, but further-more detailed studies are required.

Abundance of domestic mites in dwellings of children and adolescents with asthma in relation to environmental factors and allergy symptoms.

Exposure to house dust allergens, mainly from domestic mites, is an important cause of allergic reactions in sensitized asthmatic patients. A total of 63 dust samples were collected from 16 flats in Bytom (south Poland); in each flat a person (age 4-17 years) suffering from bronchial asthma lived with his/her family. Mite density was calculated as the number of specimens per g of dust. The results were compared with household features and the data were statistically analyzed. In total 566 mite specimens were isolated, including 526 members of the family Pyroglyphidae (93%). The dominant species were Dermatophagoides pteronyssinus (60% of the total count) and Dermatophagoides farinae (32%). Pyroglyphids were found in all mite positive samples (68%) of which 35% also contained non-pyroglyphids, including glycyphagids, cheyletids and gamasids. The results suggest associations between the density of some mite taxa (per g of dust) and the following indoor environmental factors: presence of pets, number of inhabitants, coal-stoves as a type of heating, cleaning frequency, higher relative humidity, presence of flowers and PVC windows. The severity of asthma seems to be associated with the numbers of D. farinae, total domestic mites and live mites per g of dust.

Molecular detection of tick-borne pathogens in ticks collected from pets in selected mountainous areas of Tatra County (Tatra Mountains, Poland).

The mountainous and foothill areas, in which the city of Zakopane, the capital of Tatra County, is located are characterized by continuous weather changes, lower air temperature, persistent snow cover, and poorer vegetation than in the lowlands. Ixodes ricinus and Ixodes hexagonus are vectors of tick-borne diseases and play an important role in the persistence of tick-borne diseases. The aim of the study was to determine the risk of exposure of domestic cats and dogs to the attacks of Ixodid ticks, to tick-borne infections with Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Babesia microti and Toxoplasma gondii in the city of Zakopane and the surrounding area. In 2017-2018 ticks were collected from a total of 10 domestic cats and 88 domestic dogs. Selected pathogens of tick-borne diseases were detected by PCR and nested PCR. The study material contained 119 I. ricinus and 36 I. hexagonus. The molecular examinations showed the presence of A. phagocytophilum in 3.8%, B. microti in 24.5% and T. gondii in 4.5% of the all ticks. In addition, in the study area, there is a high potential risk of tick-borne infection by B. microti, and a low potential risk of exposure to A. phagocytophilum and T. gondii infection.

Occurrence of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Babesia microti in Ixodes ricinus ticks collected from selected areas of Opolskie Province in south-west Poland.

INTRODUCTION: Ticks (Acari: Ixodida) are vectors and/or reservoirs of many pathogens, i.e. Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Babesia microti. These pathogens are ethiological agents of such diseases as Lyme borreliosis, human granulocytic anaplasmosis and human babesiosis. OBJECTIVE: The aim of the study was to evaluate the role of the Ixodes ricinus in the transmission of Borrelia burgdorferi sensu lato, Borrelia afzelii, Borrelia garinii, Borrelia burgdorferi sensu stricto, Anaplasma phagocytophilum and Babesia microti in Opolskie Province in Poland. MATERIAL AND METHODS: DNA from 222 ticks was isolated by the ammonia method. The pair of primers specific to the flagelline gene was used to detect of B. burgdorferi s. l. To detect of genospecies of this spirochete, three pairs of internal primers were used. In turn, two pairs of primers specific to the 16S rDNA gene and the 18S rRNA were used, respectively, for the detection of A. phagocytophilum and B. microti. Borrelia burgdorferi s. l., A. phagocytophilum, and B. microti were detected in 4.5%, 2.7% and 5.4% of examined ticks, respectively. RESULTS AND CONCLUSIONS: Of the ten ticks infected with B. burgdorferi s. l., B. afzelii was found in seven, undefinied genospecies in two, and mixed infection with B. afzelii and B. burgdorferi s. s. in one. The study demonstrated the potential risk of exposure of humans and animals to infections of B. burgdorferi s. l., A. phagocytophilum and B. microti in the examined area of Poland.

Two novel COL1A1 mutations in patients with osteogenesis imperfecta (OI) affect the stability of the collagen type I triple-helix.

Osteogenesis imperfecta (OI) is a bone dysplasia caused by mutations in the COL1A1 and COL1A2 genes. Although the condition has been intensely studied for over 25 years and recently over 800 novel mutations have been published, the relation between the location of mutations and clinical manifestation is poorly understood. Here we report missense mutations in COL1A1 of several OI patients. Two novel mutations were found in the D1 period. One caused a substitution of glycine 200 by valine at the N-terminus of D1 in OI type I/IV, lowering collagen stability by 50% at 34 degrees C. The other one was a substitution of valine 349 by phenylalanine at the C-terminus of D1 in OI type I, lowering collagen stability at 37.5 degrees C. Two other mutations, reported before, changed amino residues in D4. One was a lethal substitution changing glycine 866 to serine in genetically identical twins with OI type II. That mutated amino acid was near the border of D3 and D4. The second mutation changed glycine 1040 to serine located at the border of D4 and D0.4, in a proband manifesting OI type III, and lowered collagen stability at 39 degrees C (2 degrees C lower than normal). Our results confirm the hypothesis on a critical role of the D1 and D4 regions in stabilization of the collagen triple-helix. The defect in D1 seemed to produce a milder clinical type of OI, whereas the defect in the C-terminal end of collagen type caused the more severe or lethal types of OI.

Mutations in the COL1A1 and COL1A2 genes associated with osteogenesis imperfecta (OI) types I or III.

Although over 85% of osteogenesis imperfecta (OI) cases are associated with mutations in the procollagen type I genes (COL1A1 or COL1A2), no hot spots for the mutations were associated with particular clinical phenotypes. Eight patients that were studied here, diagnosed with OI by clinical standards, are from the Polish population with no ethnic background indicated. Previously unpublished mutations were found in six out of those eight patients. Genotypes for polymorphisms (Sp1 - rs1800012 and PvuII - rs412777), linked to bone formation and metabolism were determined. Mutations were found in exons 2, 22, 50 and in introns 13 and 51 of the COL1A1 gene. In COL1A2, one mutation was identified in exon 22. Deletion type mutations in COL1A1 that resulted in OI type I had no effect on collagen type I secretion, nor on its intracellular accumulation. Also, a single base substitution in I13 (c.904-9 G>T) was associated with the OI type I. The OI type III was associated with a single base change in I51 of COL1A1, possibly causing an exon skipping. Also, a missense mutation in COL1A2 changing Gly→Cys in the central part of the triple helical domain of the collagen type I molecule caused OI type III. It affected secretion of the heterotrimeric form of procollagen type I. However, no intracellular accumulation of procollagen chains could be detected. Mutation in COL1A2 affected its incorporation into procollagen type I. The results obtained shall help in genetic counseling of OI patients and provide a rational support for making informed, life important decisions by them and their families.

Surveyor nuclease detection of mutations and polymorphisms of mtDNA in children.

Mitochondrial encephalomyopathies are complex disorders with wide range of clinical manifestations. Particularly time-consuming is the identification of mutations in mitochondrial DNA. A group of 20 children with clinical manifestations of mitochondrial encephalomyopathies was selected for molecular studies. The aims were (a) to identify mutations in mtDNA isolated from muscle and (b) to verify detected mutations in DNA isolated from blood, in order to assess the utility of a Surveyor nuclease assay kit for patient screening. The most common changes found were polymorphisms, including a few missense mutations altering the amino acid sequence of mitochondrial proteins. In two boys with MELAS (i.e., mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes), a mutation A→G3243 was detected in the tRNALeu gene of mtDNA isolated from muscle and blood. In one boy, the carrier status of his mother was confirmed, based on molecular analysis of DNA isolated from blood. A method using Surveyor nuclease allows systematic screening for small mutations in mtDNA, using as its source blood of the patients and asymptomatic carriers. The method still requires confirmation studying a larger group. In some patients, the use of this method should precede and might limit indications for traumatic muscle and skin biopsy.