Development of a Large-Scale Pathogen Screening Test for the Biosafety Evaluation of Canine Mesenchymal Stem Cells.

BACKGROUND: The action of mesenchymal stem cells (MSCs) is the subject of intense research in the field of regenerative medicine, including their potential use in companion animals, such as dogs. To ensure the safety of canine MSC batches for their application in regenerative medicine, a quality control test must be conducted in accordance with Good Manufacturing Practices (GMP). Based on guidance provided by the European Medicines Agency, this study aimed to develop and validate a highly sensitive and robust, nucleic acid-based test panel for the detection of various canine pathogens. Analytical sensitivity, specificity, amplification efficiency, and linearity were evaluated to ensure robust assessment. Additionally, viable spike-in controls were used to control for optimal nucleic acid extraction. The conventional PCR-based and real-time PCR-based pathogen assays were evaluated in a real-life setting, by direct testing MSC batches. RESULTS: The established nucleic acid-based assays displayed remarkable sensitivity, detecting 100-1 copies/reaction of template DNA. They also exhibited high specificity and efficiency. Moreover, highly effective nucleic acid isolation was confirmed by the sensitive detection of spike-in controls. The detection capacity of our optimized and validated methods was determined by direct pathogen testing of nine MSC batches that displayed unusual phenotypes, such as reduced cell division or other deviating characteristics. Among these MCS batches of uncertain purity, only one tested negative for all pathogens. The direct testing of these samples yielded positive results for important canine pathogens, including tick-borne disease-associated species and viral members of the canine infectious respiratory disease complex (CIRDC). Notably, samples positive for the etiological agents responsible for enteritis (CPV), leptospirosis (Leptospira interrogans), and neosporosis (Neospora caninum) were also identified. Furthermore, we conducted biosafety evaluation of 12 MSC batches intended for therapeutic application. Eleven MSC batches were found to be free of extraneous agents, and only one tested positive for a specific pathogen, namely, canine parvovirus. CONCLUSION: In this study, we established and validated reliable, highly sensitive, and accurate nucleic acid-based testing methods for a broad spectrum of canine pathogens.

BC-Monitor: Towards a Routinely Accessible Circulating Tumor DNA-Based Tool for Real-Time Monitoring Breast Cancer Progression and Treatment Effectiveness.

Circulating tumor DNA (ctDNA) is increasingly employed in the screening, follow-up, and monitoring of the continuously evolving tumor; however, most ctDNA assays validated for clinical use cannot maintain the right balance between sensitivity, coverage, sample requirements, time, and cost. Here, we report our BC-monitor, a simple, well-balanced ctDNA diagnostic approach using a gene panel significant in breast cancer and an optimized multiplex PCR-based NGS protocol capable of identifying allele variant frequencies below 1% in cell-free plasma DNA. We monitored a cohort of 45 breast cancer patients prospectively enrolled into our study receiving neoadjuvant chemotherapy or endocrine therapy or palliative therapy for metastatic diseases. Their tumor mutation status was examined in the archived tumor samples and plasma samples collected before and continuously during therapy. Traceable mutations of the used 38-plex NGS assay were found in approximately two-thirds of the patients. Importantly, we detected new pathogenic variants in follow-up plasma samples that were not detected in the primary tumor and baseline plasma samples. We proved that the BC-monitor can pre-indicate disease progression four-six months earlier than conventional methods. Our study highlights the need for well-designed ctDNA monitoring during treatment and follow-up, integrated into a real-time treatment assessment, which could provide information on the active tumor DNA released into the blood.

Genetic analysis of the SOD1 and C9ORF72 genes in Hungarian patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the death of motor neurons. To date, more than 20 genes have been implicated in ALS, and of these, the 2 most frequently mutated are the superoxide dismutase 1 (SOD1) gene and the chromosome 9 open reading frame 72 (C9ORF72) gene. In this study, we aimed to investigate the contribution of these 2 Mendelian genes to the development of the disease in Hungarian ALS patients (n = 66). Direct sequencing of the SOD1 gene revealed a novel (p.Lys91ArgfsTer8) and 3 recurrent heterozygous mutations (p.Val14Met, p.Asp90Ala, and p.Leu144Phe) in 5 patients. The novel p.Lys91ArgfsTer8 mutation led to a frameshift causing the addition of 8 new amino acids, including a premature stop codon at position 99. The GGGGCC hexanucleotide repeat expansion of the C9ORF72 gene was present in 1 ALS patient. This study represents the first genetic analysis of 2 major ALS causative genes in a cohort of Hungarian ALS patients and contributes to the further understanding of the genetic and phenotypic diversity of ALS.

Different phenotypes in identical twins with cerebrotendinous xanthomatosis: case series.

Cerebrotendinous xanthomatosis (CTX) is a rare, genetically determined error of metabolism. The characteristic clinical symptoms are diarrhea, juvenile cataracts, tendon xanthomas and neuropsychiatric alterations. The aim of this study is to present a pair of identical adult twins with considerable differences in the severity of phenotype. With regards to neuropsychiatric symptoms, the predominant features were severe Parkinsonism and moderate cognitive dysfunctions in the more-affected individual, whereas these alterations in the less-affected patient were only very mild and mild, respectively. The characteristic increase in the concentrations of serum cholestanol and the lesion volumes in dentate nuclei in the brain assessed with magnetic resonance imaging were quite similar in both cases. The lifestyle conditions, including eating habits of the twin pair, were quite similar as well; therefore, currently unknown genetic modifiers or certain epigenetic factors may be responsible for the differences in severity of phenotype. This case series serves as the first description of an identical twin pair with CTX presenting heterogeneous clinical features.

Genetic structure of the early Hungarian conquerors inferred from mtDNA haplotypes and Y-chromosome haplogroups in a small cemetery.

We applied ancient DNA methods to shed light on the origin of ancient Hungarians and their relation to modern populations. Hungarians moved into the Carpathian Basin from the Eurasian Pontic steppes in the year 895 AD as a confederation of seven tribes, but their further origin remains obscure. Here, we present 17 mtDNA haplotypes and four Y-chromosome haplogroups, which portray the genetic composition of an entire small cemetery of the first generation Hungarians. Using novel algorithms to compare these mitochondrial DNA haplogroups with other ancient and modern Eurasian data, we revealed that a significant portion of the Hungarians probably originated from a long ago consolidated gene pool in Central Asia-South Siberia, which still persists in modern Hungarians. Another genetic layer of the early Hungarians was obtained during their westward migrations by admixing with various populations of European origin, and an important component of these was derived from the Caucasus region. Most of the modern populations, which are genetically closest relatives of ancient Hungarians, today speak non-Indo-European languages. Our results contribute to our understanding of the peopling of Europe by providing ancient DNA data from a still genetically poorly studied period of medieval human migrations.

Comparison of lactase persistence polymorphism in ancient and present-day Hungarian populations.

The prevalence of adult-type hypolactasia varies ethnically and geographically among populations. A C/T-13910 single nucleotide polymorphism (SNP) upstream of the lactase gene is known to be associated with lactase non-persistence in Europeans. The aim of this study was to determine the prevalence of lactase persistent and non-persistent genotypes in current Hungarian-speaking populations and in ancient bone samples of classical conquerors and commoners from the 10th-11th centuries from the Carpathian basin; 181 present-day Hungarian, 65 present-day Sekler, and 23 ancient samples were successfully genotyped for the C/T-13910 SNP by the dCAPS PCR-RFLP method. Additional mitochondrial DNA testing was also carried out. In ancient Hungarians, the T-13910 allele was present only in 11% of the population, and exclusively in commoners of European mitochondrial haplogroups who may have been of pre-Hungarian indigenous ancestry. This is despite animal domestication and dairy products having been introduced into the Carpathian basin early in the Neolithic Age. This anomaly may be explained by the Hungarian use of fermented milk products, their greater consumption of ruminant meat than milk, cultural differences, or by their having other lactase-regulating genetic polymorphisms than C/T-13910. The low prevalence of lactase persistence provides additional information on the Asian origin of Hungarians. Present-day Hungarians have been assimilated with the surrounding European populations, since they do not differ significantly from the neighboring populations in their possession of mtDNA and C/T-13910 variants.

Comparison of maternal lineage and biogeographic analyses of ancient and modern Hungarian populations.

The Hungarian language belongs to the Finno-Ugric branch of the Uralic family, but Hungarian speakers have been living in Central Europe for more than 1000 years, surrounded by speakers of unrelated Indo-European languages. In order to study the continuity in maternal lineage between ancient and modern Hungarian populations, polymorphisms in the HVSI and protein coding regions of mitochondrial DNA sequences of 27 ancient samples (10th-11th centuries), 101 modern Hungarian, and 76 modern Hungarian-speaking Sekler samples from Transylvania were analyzed. The data were compared with sequences derived from 57 European and Asian populations, including Finno-Ugric populations, and statistical analyses were performed to investigate their genetic relationships. Only 2 of 27 ancient Hungarian samples are unambiguously Asian: the rest belong to one of the western Eurasian haplogroups, but some Asian affinities, and the genetic effect of populations who came into contact with ancient Hungarians during their migrations are seen. Strong differences appear when the ancient Hungarian samples are analyzed according to apparent social status, as judged by grave goods. Commoners show a predominance of mtDNA haplotypes and haplogroups (H, R, T), common in west Eurasia, while high-status individuals, presumably conquering Hungarians, show a more heterogeneous haplogroup distribution, with haplogroups (N1a, X) which are present at very low frequencies in modern worldwide populations and are absent in recent Hungarian and Sekler populations. Modern Hungarian-speaking populations seem to be specifically European. Our findings demonstrate that significant genetic differences exist between the ancient and recent Hungarian-speaking populations, and no genetic continuity is seen.

Mitochondrial DNA control region analysis of a late Neolithic aurochs (Bos primigenius Boj. 1827) from the Carpathian Basin.

Bos primigenius, the wild aurochs is believed to be the ancestor of European domestic cattle, Bos taurus. The geography and climate of the Great Hungarian Plain were well suited for these large grazing animals in the Late Neolithic. Till now, there are just a few aurochs mtDNA fragments available from two geographically restricted area, the British Isles and Italy. To increase our knowledge about the genetics of the European aurochsen livestock, and to investigate the phylogenetic position of a late Neolithic aurochs, excavated from the Carpathian Basin, mitochondrial DNA was extracted from a fragment of corpus mandibulae using ancient-DNA techniques and a portion of mitochondrial hypervariable region was amplified by PCR. The resulting sequence was aligned with GenBank sequences of 11 aurochsen. Our new sequence is identical with the sequence of two British aurochs. The 6000-year-old Hungarian aurochs shows a mtDNA sequence pattern, that occurs only among 6-12,000-year-old North European aurochsen, and it does not occur among modern, domesticated cattle.

Mitochondrial DNA of ancient Cumanians: culturally Asian steppe nomadic immigrants with substantially more western Eurasian mitochondrial DNA lineages.

The Cumanians were originally Asian pastoral nomads who in the 13th century migrated to Hungary. We have examined mitochondrial DNA from members of the earliest Cumanian population in Hungary from two archeologically well-documented excavations and from 74 modern Hungarians from different rural locations in Hungary. Haplogroups were defined based on HVS I sequences and examinations of haplogroup-associated polymorphic sites of the protein coding region and of HVS II. To exclude contamination, some ancient DNA samples were cloned. A database was created from previously published mtDNA HVS I sequences (representing 2,615 individuals from different Asian and European populations) and 74 modem Hungarian sequences from the present study. This database was used to determine the relationships between the ancient Cumanians, modern Hungarians, and Eurasian populations and to estimate the genetic distances between these populations. We attempted to deduce the genetic trace of the migration of Cumanians. This study is the first ancient DNA characterization of an eastern pastoral nomad population that migrated into Europe. The results indicate that, while still possessing a Central Asian steppe culture, the Cumanians received a large admixture of maternal genes from more westerly populations before arriving in Hungary. A similar dilution of genetic, but not cultural, factors may have accompanied the settlement of other Asian nomads in Europe.