Deciphering the role of a SINE-VNTR-Alu retrotransposon polymorphism as a biomarker of Parkinson's disease progression.

SINE-VNTR-Alu (SVA) retrotransposons are transposable elements which represent a source of genetic variation. We previously demonstrated that the presence/absence of a human-specific SVA, termed SVA_67, correlated with the progression of Parkinson's disease (PD). In the present study, we demonstrate that SVA_67 acts as expression quantitative trait loci, thereby exhibiting a strong regulatory effect across the genome using whole genome and transcriptomic data from the Parkinson's progression markers initiative cohort. We further show that SVA_67 is polymorphic for its variable number tandem repeat domain which correlates with both regulatory properties in a luciferase reporter gene assay in vitro and differential expression of multiple genes in vivo. Additionally, this variation's utility as a biomarker is reflected in a correlation with a number of PD progression markers. These experiments highlight the plethora of transcriptomic and phenotypic changes associated with SVA_67 polymorphism which should be considered when investigating the missing heritability of neurodegenerative diseases.

Unravelling Chlamydia trachomatis diversity in Amhara, Ethiopia: MLVA-ompA sequencing as a molecular typing tool for trachoma.

Trachoma is the leading infectious cause of blindness worldwide and is now largely confined to around 40 low- and middle-income countries. It is caused by Chlamydia trachomatis (Ct), a contagious intracellular bacterium. The World Health Organization recommends mass drug administration (MDA) with azithromycin for treatment and control of ocular Ct infections, alongside improving facial cleanliness and environmental conditions to reduce transmission. To understand the molecular epidemiology of trachoma, especially in the context of MDA and transmission dynamics, the identification of Ct genotypes could be useful. While many studies have used the Ct major outer membrane protein gene (ompA) for genotyping, it has limitations. Our study applies a typing system novel to trachoma, Multiple Loci Variable Number Tandem Repeat Analysis combined with ompA (MLVA-ompA). Ocular swabs were collected post-MDA from four trachoma-endemic zones in Ethiopia between 2011-2017. DNA from 300 children with high Ct polymerase chain reaction (PCR) loads was typed using MLVA-ompA, utilizing 3 variable number tandem repeat (VNTR) loci within the Ct genome. Results show that MLVA-ompA exhibited high discriminatory power (0.981) surpassing the recommended threshold for epidemiological studies. We identified 87 MLVA-ompA variants across 26 districts. No significant associations were found between variants and clinical signs or chlamydial load. Notably, overall Ct diversity significantly decreased after additional MDA rounds, with a higher proportion of serovar A post-MDA. Despite challenges in sequencing one VNTR locus (CT1299), MLVA-ompA demonstrated cost-effectiveness and efficiency relative to whole genome sequencing, providing valuable information for trachoma control programs on local epidemiology. The findings suggest the potential of MLVA-ompA as a reliable tool for typing ocular Ct and understanding transmission dynamics, aiding in the development of targeted interventions for trachoma control.

Exploring SVA Insertion Polymorphisms in Shaping Differential Gene Expressions in the Central Nervous System.

Transposable elements (TEs) are repetitive elements which make up around 45% of the human genome. A class of TEs, known as SINE-VNTR-Alu (SVA), demonstrate the capacity to mobilise throughout the genome, resulting in SVA polymorphisms for their presence or absence within the population. Although studies have previously highlighted the involvement of TEs within neurodegenerative diseases, such as Parkinson's disease and amyotrophic lateral sclerosis (ALS), the exact mechanism has yet to be identified. In this study, we used whole-genome sequencing and RNA sequencing data of ALS patients and healthy controls from the New York Genome Centre ALS Consortium to elucidate the influence of reference SVA elements on gene expressions genome-wide within central nervous system (CNS) tissues. To investigate this, we applied a matrix expression quantitative trait loci analysis and demonstrate that reference SVA insertion polymorphisms can significantly modulate the expression of numerous genes, preferentially in the trans position and in a tissue-specific manner. We also highlight that SVAs significantly regulate mitochondrial genes as well as genes within the HLA and MAPT loci, previously associated within neurodegenerative diseases. In conclusion, this study continues to bring to light the effects of polymorphic SVAs on gene regulation and further highlights the importance of TEs within disease pathology.

Common single-base insertions in the VNTR of the carboxyl ester lipase (CEL) gene are benign and also likely to arise somatically in the exocrine pancreas.

The CEL gene encodes carboxyl ester lipase, a pancreatic digestive enzyme. CEL is extremely polymorphic due to a variable number tandem repeat (VNTR) located in the last exon. Single-base deletions within this VNTR cause the inherited disorder MODY8, whereas little is known about VNTR single-base insertions in pancreatic disease. We therefore mapped CEL insertion variants (CEL-INS) in 200 Norwegian patients with pancreatic neoplastic disorders. Twenty-eight samples (14.0%) carried CEL-INS alleles. Most common were insertions in repeat 9 (9.5%), which always associated with a VNTR length of 13 repeats. The combined INS allele frequency (0.078) was similar to that observed in a control material of 416 subjects (0.075). We performed functional testing in HEK293T cells of a set of CEL-INS variants, in which the insertion site varied from the first to the 12th VNTR repeat. Lipase activity showed little difference among the variants. However, CEL-INS variants with insertions occurring in the most proximal repeats led to protein aggregation and endoplasmic reticulum stress, which upregulated the unfolded protein response. Moreover, by using a CEL-INS-specific antibody, we observed patchy signals in pancreatic tissue from humans without any CEL-INS variant in the germline. Similar pancreatic staining was seen in knock-in mice expressing the most common human CEL VNTR with 16 repeats. CEL-INS proteins may therefore be constantly produced from somatic events in the normal pancreatic parenchyma. This observation along with the high population frequency of CEL-INS alleles strongly suggests that these variants are benign, with a possible exception for insertions in VNTR repeats 1-4.

Laboratory and field validation of the recombinase polymerase amplification assay targeting the Schistosoma mansoni mitochondrial minisatellite region (SmMIT-RPA) for snail xenomonitoring for schistosomiasis.

Improvements in diagnostics for schistosomiasis in both humans and snail hosts are priorities to be able to reach the World Health Organization (WHO) goal of eliminating the disease as a public health problem by 2030. In this context, molecular isothermal amplification tests, such as Recombinase Polymerase Amplification (RPA), are promising for use in endemic areas at the point-of-need for their accuracy, robustness, simplicity, and time-effectiveness. The developed recombinase polymerase amplification assay targeting the Schistosoma mansoni mitochondrial minisatellite region (SmMIT-RPA) was used to detect S. mansoni DNA from both laboratory and field Biomphalaria snails. Laboratory snails were experimentally infected and used at one, seven, and 28 days post-exposure (dpe) to 10 S. mansoni miracidia to provide samples in the early pre-patent infection stage. Field samples of Biomphalaria spp. were collected from the Mucuri Valley and Jequitinhonha Valley regions in the state of Minas Gerais, Brazil, which are endemic for S. mansoni. The sensitivity and specificity of the SmMIT-RPA assay were analysed and compared with existing loop-mediated isothermal amplification (LAMP), PCR-based methods, parasitological examination of the snails, and nucleotide sequencing. The SmMIT-RPA assay was able to detect S. mansoni DNA in the experimentally infected Biomphalaria glabrata as early as one dpe to 10 miracidia. It also detected S. mansoni infections (55.5% prevalence) in the field samples with the highest accuracy (100% sensitivity and specificity) compared with the other molecular tests used as the reference. Results from this study indicate that the SmMIT-RPA assay is a good alternative test to be used for snail xenomonitoring of S. mansoni due to its high sensitivity, accuracy, and the possibility of detecting early pre-patent infection. Its simplicity and portability also make it a suitable methodology in low-resource settings.

Population structure of Mycobacterium tuberculosis in El Oro: A first insight into Ecuador-Peru tuberculosis transmission.

BACKGROUND: Tuberculosis (TB) is a major public health concern in Ecuador and Peru, both settings of high burden of drug resistance TB. Molecular epidemiology tools are important to understand the transmission dynamics of Mycobacterium tuberculosis Complex (MTBC) and to track active transmission clusters of regional importance. This study is the first to address the transmission of TB between Peru and Ecuador through the population structure of MTBC lineages circulating in the Ecuadorian border province of "El Oro". METHODS: A total number of 56 MTBC strains from this province for years 2012-2015 were included in the study and analyzed by 24-loci MIRU-VNTR and spoligotyping. RESULTS: Genotyping revealed a high degree of diversity for MTBC in "El Oro", without active transmission clusters. MTBC L4 was predominant, with less than 2% of strains belonging to MTBC L2-Beijing. CONCLUSIONS: These results may suggest that TB dynamics in this rural and semi-urban area would not be linked to highly transmitted strains like MTBC L2-Beijing from Peru, but related to TB relapse; although further studies with larger MTBC cultures collection from recent years are needed. Nevertheless, we recommend to reinforce TB surveillance programs in remote rural settings and border regions in Ecuador.

The role of the genetic influence of DRD4 in Chinese adults in the context of the choice of tourist attractions.

The aim of this work was to analyse the effect of tandem repetitions in exon III of the DRD4 gene on the features of human decision-making in a model of choosing tourist attractions by adult residents of China. The study included 380 subjects: 162 (42.6%) men and 218 (57.4%) women. The mean age of the subjects was 31.7 ± 3.32 years. As a result of the survey of subjects, 5 groups of motivations for choosing tourist attractions were identified, and the frequency of their use, including the identified combinations, was determined. Using the genotyping method, the frequency of DRD4 subtypes among the subjects was determined, and their relationship with the indicated attraction selection groups was studied. It has been established that there is a significant dependence of the frequency of choosing the attractors 'relaxation', 'desire for novelty' and 'self-realization' and their combinations on the frequency of occurrence of the DRD4 2R, 4R and 5R+ subtypes in the study groups. A conclusion was made about the possible mechanism of the influence of manifestations of DRD4 subtypes on the choice of tourist attractors by implementing the neurophysiological influence of the genome on reducing the sensitivity of brain receptors to dopamine, which stimulates behaviours that compensate for the need for additional emotional influences. This work complements the existing knowledge about the impact of human innate properties on the characteristics of his behaviour and possible patterns of influence of human genotype variability on decision-making and suggests further possible directions of research in this area.

Association of the neonatal Fc receptor promoter variable number of tandem repeat polymorphism with immunoglobulin response in patients with chronic inflammatory demyelinating polyneuropathy.

BACKGROUND AND PURPOSE: Chronic inflammatory demyelinating polyneuropathy (CIDP) is an autoimmune disease with humoral and cellular autoimmunity causing demyelination of peripheral nerves, commonly treated with intravenous immunoglobulins (IVIg). The neonatal Fc receptor (FcRn), encoded by the FCGRT gene, prevents the degradation of immunoglobulin G (IgG) by recycling circulating IgG. A variable number of tandem repeat (VNTR) polymorphism in the promoter region of the FCGRT gene is associated with different expression levels of mRNA and protein. Thus, patients with genotypes associated with relatively low FcRn expression may show a poorer treatment response to IVIg due to increased IVIg degradation. METHODS: VNTR genotypes were analyzed in 144 patients with CIDP. Patients' clinical data, including neurological scores and treatment data, were collected as part of the Immune-Mediated Neuropathies Biobank registry. RESULTS: Most patients (n = 124, 86%) were VNTR 3/3 homozygotes, and 20 patients (14%) were VNTR 2/3 heterozygotes. Both VNTR 3/3 and VNTR 2/3 genotype groups showed no difference in clinical disability and immunoglobulin dosage. However, patients with a VNTR 2 allele were more likely to receive subcutaneous immunoglobulins (SCIg) than patients homozygous for the VNTR 3 allele (25% vs. 9.7%, p = 0.02) and were more likely to receive second-line therapy (75% vs. 54%, p = 0.05). CONCLUSIONS: The VNTR 2/3 genotype is associated with the administration of SCIg, possibly reflecting a greater benefit from SCIg due to more constant immunoglobulin levels without lower IVIg levels between the treatment circles. Also, the greater need for second-line treatment in VNTR 2/3 patients could be an indirect sign of a lower response to immunoglobulins.

Association of tyrosine hydroxylase 01 (TH01) microsatellite and insulin gene (INS) variable number of tandem repeat (VNTR) with type 2 diabetes and fasting insulin secretion in Mexican population.

PURPOSE: A variable number of tandem repeats (VNTR) in the insulin gene (INS) control region may be involved in type 2 diabetes (T2D). The TH01 microsatellite is near INS and may regulate it. We investigated whether the TH01 microsatellite and INS VNTR, assessed via the surrogate marker single nucleotide polymorphism rs689, are associated with T2D and serum insulin levels in a Mexican population. METHODS: We analyzed a main case-control study (n = 1986) that used univariate and multivariate logistic regression models to calculate the risk conferred by TH01 and rs689 loci for T2D development; rs689 results were replicated in other case-control (n = 1188) and cross-sectional (n = 1914) studies. RESULTS: TH01 alleles 6, 8, 9, and 9.3 and allele A of rs689 were independently associated with T2D, with differences between sex and age at diagnosis. TH01 alleles with ≥ 8 repeats conferred an increased risk for T2D in males compared with ≤ 7 repeats (odds ratio, ≥ 1.46; 95% confidence interval, 1.1-1.95). In females, larger alleles conferred a 1.5-fold higher risk for T2D when diagnosed ≥ 46 years but conferred protection when diagnosed ≤ 45 years. Similarly, rs689 allele A was associated with T2D in these groups. In males, larger TH01 alleles and the rs689 A allele were associated with a significant decrease in median fasting plasma insulin concentration with age in T2D cases; the reverse occurred in controls. CONCLUSION: Larger TH01 alleles and rs689 A allele may potentiate insulin synthesis in males without T2D, a process disabled in those with T2D.

Evaluation of a circadian rhythm gene (PER3) VNTR variant in Turkish athletes.

OBJECTIVE: Circadian rhythmicity has been shown to contribute to the regulation of key physiological and cognitive processes related to performance. The period homolog 3 (PER3) is expressed in a circadian pattern in the suprachiasmatic nucleus. Therefore, in this study, we aimed to evaluate the role of the variable tandem repeat (VNTR) variant of the PER3 gene in athletic performance in the Turkish population. METHODS: This study included 223 subjects, which consisted of 123 athletes and 100 sedentary controls. Blood samples were drawn from all subjects. DNA was extracted from whole-blood samples. The PER3 VNTR variant was genotyped using the polymerase chain reaction-restriction method (PCR). The results of the analyses were evaluated for statistical significance. RESULTS: The mean ages of athletes and controls were 22 ± 2.814 and 23 ± 3.561, respectively. Endurance athletes in the group were 21.1%, and sprint athletes were 78.9%. There was no statistical significance in terms of PER3 VNTR genotype distribution or allele frequency. In the recessive model, a statistically significant association was observed when the athletes were compared with the controls according to 4/4 + 4/5 versus 5/5 genotype (p = 0.020). CONCLUSION: In this case-control study, for the first time in our country, we obtained findings suggesting that the PER3 VNTR variant may affect sports performance in the Turkish population. Results need to be replicated in different ethnic and larger samples.

DRD4 VNTR 4/4 homozygosity as a genetic biomarker for treatment selection in patients with schizophrenia.

OBJECTIVE: There seems to be an association between the DRD4 48-bp VNTR polymorphisms and antipsychotic treatment response, but there is a rare reference to confirm this finding. Hence, the present study tried to investigate the association between DRD4 48-bp VNTR polymorphisms and the treatment response of antipsychotics in patients with schizophrenia in Taiwan, using a propensity score matching (PSM) method. METHODS: A total of 882 participants were enrolled in this study and completed informed consent, research questionnaires, including demographic information and the revised Chinese version Beliefs about Voices Questionnaire, and blood sampling. For descreasing of the selection bias and confounding variables, the PSM nearest neighbor matching method was used to select 765 paitents with schizophrenia (ratio of 1:8 between 85 persistent auditory hallucination and 680 controls) with matched and controlled the age and gender. RESULTS: Schizophrenia patients with DRD4 4 R homozygosity had a lower rate of good antipsychotic treatment response than the other DRD4 genotype carriers (DRD4 non-4/4). Among those 4 R homozygosity carriers, 60 cases of 503 (11.9%) retain persistent auditory hallucinations. Furthermore, this subgroup of patients is accounted for up to 70.6% of cases with poor neuroleptic treatment response. CONCLUSIONS: A poor treatment outcome for patients with the 4 R homozygosity had presented,that comparing with those DRD non-4/4 genotype carriers. DRD4 VNTR 4 R homozygosity could be a genetic biomarker to predict poor antipsychotic treatment response in schizophrenia. Patients with DRD 4/4 probably receive novel antipsychotic medications preferentially or in combination with alternative therapy, such as psychotherapy or milieu therapy.

Molecular epidemiology and drug sensitivity of Mycobacterium tuberculosis in homeless individuals in the Addis Ababa city, Ethiopia.

Although homeless segment of the society could be the hotspots for tuberculosis (TB) transmission, there is little data on TB in homeless individuals in Ethiopia. The objective of this study was to investigate the molecular epidemiology and drug sensitivity of Mycobacterium tuberculosis (M. tuberculosis) isolated from homeless individuals in Addis Ababa, Ethiopia. The study was conducted on 59 M. tuberculosis isolates, which were recovered by the clinical screening of 5600 homeless individuals and bacteriological examination of 641 individuals with symptoms of pulmonary tuberculosis (PTB). Region of difference-9 (RD9) based polymerase-chain reaction (PCR), Spoligotyping and 24-loci Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) typing were used for genotyping of the isolates. In addition, drug sensitivity test was performed on the isolates using BD Bactec Mycobacterial Growth Inhibition Tube (MGIT) 960. Fifty-eight of the 59 isolates were positive by spoligotyping and spoligotyping International type (SIT) 53, SIT 37, and SIT 149 were the dominant spoligotypes; each consisting of 19%, 15.5%, and10.3% of the isolates, respectively. The majority of the isolates (89.7%) were members of the Euro-American (EA) major lineage. MIRU-VNTR identified Ethiopia_3, Delhi/CAS, Ethiopia_2, TUR, X-type, Ethiopia_H37Rv-like strain, Haarlem and Latin-American Mediterranean (LAM) sub lineages. The proportion of clustering was 77.6% (45/58) in spoligotyping while it was 39.7% (23/58) in 24-loci MIRU-VNTR typing. Furthermore, the proportion of clustering was significantly lowered to 10.3% (6/58) when a combination of spoligotyping and 24-loci MIRU-VNTRplus was used. The recent transmission index (RTI) recorded by spoligotyping, 24-loci MIRU-VNTR typing, and a combination of the two genotyping methods were 58.6%, 27.6% and 5.2%, respectively. Young age and living in groups were significantly associated with strain clustering (P < 0.05). The drug sensitivity test (DST) result showed 8.9% (4/58) of the isolates were resistant to one or more first line ant-TB drugs; but multidrug resistant isolate was not detected. Clustering and RTI could suggest the transmission of TB in the homeless individuals, which could suggest a similar pattern of transmission between homeless individuals and the general population. Hence, the TB control program should consider homeless individuals during the implementation of TB control program.

THE ROLE OF HEREDITARY PREDISPOSITION (POLYMORPHIC MARKERS OF GLUTATHIONE-S-TRANSFERASE, CATALASE, ENDOTHELIAL NITROGEN OXIDE SYNTHASE GENES) AND SOME ADVERSE ENVIRONMENTAL FACTORS IN THE DEVELOPMENT OF BRONCHO-OBSTRUCTIVE PATHOLOGY IN CHILDREN LIVING IN RADIOACTIVELY CONTAMINATED AREAS. / РОЛЬ СПАДКОВОЇ СХИЛЬНОСТІ (ПОЛІМОРФНИХ МАРКЕРІВ ГЕНІВ ГЛУТАТІОН-S-ТРАНСФЕРАЗ, КАТАЛАЗИ, ЕНДОТЕЛІАЛЬНОЇ СИНТАЗИ ОКСИДУ АЗОТУ) ТА ДЕЯКИХ НЕСПРИЯТЛИВИХ ЧИННИКІВ ДОВКІЛЛЯ У ВИНИКНЕННІ БРОНХООБСТРУКТИВНОЇ ПАТОЛОГІЇ У ДІТЕЙ, МЕШКАНЦІВ РАДІОАКТИВНО ЗАБРУДНЕНИХ ТЕРИТОРІЙ.

OBJECTIVE: summarizing the results of many years of research by the authors on the influence of gene polymorphisms encoding xenobiotic biotransformation enzymes (GSTТ1, GSTM1, GSTР1), antioxidant protection (С^262Т of the catalase gene), endothelial nitric oxide synthase (4a/4b VNTR polymorphism of the eNOS gene), and some environmental factors on the occurrence of broncho-obstructive disorders and the development of bronchial asthma in children, residents of radioactively contaminated areas. MATERIALS AND METHODS: The examined school-aged children were residents of radioactively RCA who had no clinical signs of respiratory pathology. Deletion polymorphism of catalase gene (CAT C^262T), polymorphism of glutathione-S-transferase gene (GSTТ1, GSTM1, GSTР1) and the polymorphism in the 4th intron (4a/4b) of the eNOS gene were studied in the molecular genetics laboratory of the State Institution «Reference Center for Molecular Diagnostics of Public Health Ministry of Ukraine¼. Molecular genetic studies were performed by polymerase chain reaction. The study of the ventilation lung capacity was carried out by the method of computer spirometry based on the data of the «flow-volume¼ loop analysis. A pharmacological inhalation test with a bronchodilator drug which affects the ß2-adrenergic receptors of the lungs was used to detect early changes in the ventilatory lung capacity - bronchial hyperreactivity. RESULTS AND CONCLUSIONS: One of the leading mechanisms, due to which the implementation of hereditary predisposition to bronchial asthma in children living in radioactively contaminated areas is the polymorphism of certain genes of glutathione-S-transferase, catalase, endothelial nitric oxide synthase. With such polymorphic variants of the GST genes, isoforms of enzymes with reduced activity are produced, which limits their ability to effectively neutralize free radicals, which are formed in excess when free radical oxidation processes are activated due to the constant intake of radionuclides with a long half-life into the body of children. Unfavorable factors that increase the risk of developing broncho-obstructive disorders and the likelihood of their implementation in the form of bronchial asthma in children, residents of radioactively contaminated areas, have been identified. It has been established that among them the leading role is played by hereditary predisposition to this disease. On the part of the child, such negative factors were unfavorable conditions of intrauterine development, the presence of signs of exudative-catarrhal diathesis, manifestations of allergies and frequent respiratory diseases from the first months of life.

Genotyping methods and their contributions to the study of tuberculosis dynamic in Latin America.

INTRODUCTION: Mycobacterium tuberculosis genotyping has impacted evolutionary studies worldwide. Nonetheless, its application and the knowledge generated depend on the genetic marker evaluated and the detection technologies that have evolved over the years. Here we describe the timeline of main genotypic methods related to M. tuberculosis in Latin America and the main findings obtained. METHODOLOGY: Systematic searches through the PubMed database were performed from 1993 to May 2021. A total of 345 articles met the inclusion criteria and were selected. RESULTS: Spacer oligonucleotide typing (spoligotyping) was the most widely used method in Latin America, with decreasing use in parallel with increasing use of mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) and whole genome sequencing (WGS). Among the countries, Brazil, Mexico, and Argentina had the most publications, and a considerable part of the articles were in collaboration with Latin American or non-Latin American institutions; a small proportion of studies needed partnerships to perform the genotypic methods. The genotypic methods allowed the identification of M. tuberculosis genotypes with greater capacity for clonal expansion and revealed the predominance of the Euro-American lineage in Latin America. There was a notable presence of the Beijing family in Peru and Colombia. CONCLUSIONS: The data obtained demonstrated the importance of expanding collaborative networks of tuberculosis (TB) research groups to countries with low productivity in this area, the commitment of the few Latin American countries to advance TB research, as well as the inestimable value of building a Latin America database, considering ease of population mobility between countries.

Mitogenome sequences of domestic cats demonstrate lineage expansions and dynamic mutation processes in a mitochondrial minisatellite.

BACKGROUND: As a population genetic tool, mitochondrial DNA is commonly divided into the ~ 1-kb control region (CR), in which single nucleotide variant (SNV) diversity is relatively high, and the coding region, in which selective constraint is greater and diversity lower, but which provides an informative phylogeny. In some species, the CR contains variable tandemly repeated sequences that are understudied due to heteroplasmy. Domestic cats (Felis catus) have a recent origin and therefore traditional CR-based analysis of populations yields only a small number of haplotypes. RESULTS: To increase resolution we used Nanopore sequencing to analyse 119 cat mitogenomes via a long-amplicon approach. This greatly improves discrimination (from 15 to 87 distinct haplotypes in our dataset) and defines a phylogeny showing similar starlike topologies within all major clades (haplogroups), likely reflecting post-domestication expansion. We sequenced RS2, a CR tandem array of 80-bp repeat units, placing RS2 array structures within the phylogeny and increasing overall haplotype diversity. Repeat number varies between 3 and 12 (median: 4) with over 30 different repeat unit types differing largely by SNVs. Five SNVs show evidence of independent recurrence within the phylogeny, and seven are involved in at least 11 instances of rapid spread along repeat arrays within haplogroups. CONCLUSIONS: In defining mitogenome variation our study provides key information for the forensic genetic analysis of cat hair evidence, and for the first time a phylogenetically informed picture of tandem repeat variation that reveals remarkably dynamic mutation processes at work in the mitochondrion.

Development of molecular assays for the analysis of genetic relationships of Mycoplasma iowae.

Mycoplasma iowae is a worldwide spread and economically important avian pathogen that mostly infects turkeys. Currently, multi-locus sequence typing (MLST) serves as the gold standard method for strain identification in M. iowae. However, additional robust genotyping methods are required to effectively monitor M. iowae infections and conduct epidemiological investigations. The first aim of this study was to develop genotyping assays with high resolution, that specifically target M. iowae, namely a multiple-locus variable number of tandem-repeats analysis (MLVA) and a core genome multi-locus sequence typing (cgMLST) schema. The second aim was the determination of relationships among a diverse selection of M. iowae strains and clinical isolates with a previous and the newly developed assays. The MLVA was designed based on the analyses of tandem-repeat (TR) regions in the six serotype reference strains (I, J, K, N, Q and R). The cgMLST schema was developed based on the coding sequences (CDSs) common in 95% of the examined 99 isolates. The samples were submitted for a previously published MLST assay for comparison with the developed methods. Out of 94 TR regions identified, 17 alleles were selected for further evaluation by PCR. Finally, seven alleles were chosen to establish the MLVA assay. Additionally, whole genome sequence analyses identified a total of 676 CDSs shared by 95% of the isolates, all of which were included into the developed cgMLST schema. The MLVA discriminated 19 distinct genotypes (GT), while with the cgMLST assay 79 sequence types (ST) could be determined with Simpson's diversity indices of 0.810 (MLVA) and 0.989 (cgMLST). The applied assays consistently identified the same main clusters among the diverse selection of isolates, thereby demonstrating their suitability for various genetic analyses and their ability to yield congruent results.

Insight into the genetic diversity of Mycobacterium bovis isolated from cattle in Malawi.

We describe the genetic diversity and phylogenetic relationships of Mycobacterium bovis, isolated from cattle in Malawi. Deletion analysis, spoligotyping, and MIRU-VNTR typing were used to genotype the isolates. Combined with a larger dataset from neighboring countries, the overall M. bovis diversity in Southern Africa was contextualized. From the southern and northern regions of Malawi, 24 isolates were confirmed as M. bovis. We pooled data for the central region (60 isolates) from our recent publication to conceptualize the genetic and phylogenetic relationships of M. bovis in Malawi. European 1 was the dominant M. bovis clonal complex, with 10 unique spoligotype patterns, and SB0131 was ubiquitous. High genetic diversity, a low clustering rate, and many singletons, coupled with a low mutation transmission index, infer a low level of recent transmission, and suggest an endemic status of bovine tuberculosis (bTB) in Malawi. M. bovis isolates from Zambia, Mozambique, and South Africa were genetically related to Malawian isolates, whereas Tanzanian isolates were distantly related. The diversity and phylogenetic analysis suggest earlier introductions and maintenance of M. bovis by constant reinfection from reservoir animals. These findings are fundamental to understanding the source and route of infection in order to establish alternative management strategies for bTB.

The landscape of human SVA retrotransposons.

SINE-VNTR-Alu (SVA) retrotransposons are evolutionarily young and still-active transposable elements (TEs) in the human genome. Several pathogenic SVA insertions have been identified that directly mutate host genes to cause neurodegenerative and other types of diseases. However, due to their sequence heterogeneity and complex structures as well as limitations in sequencing techniques and analysis, SVA insertions have been less well studied compared to other mobile element insertions. Here, we identified polymorphic SVA insertions from 3646 whole-genome sequencing (WGS) samples of >150 diverse populations and constructed a polymorphic SVA insertion reference catalog. Using 20 long-read samples, we also assembled reference and polymorphic SVA sequences and characterized the internal hexamer/variable-number-tandem-repeat (VNTR) expansions as well as differing SVA activity for SVA subfamilies and human populations. In addition, we developed a module to annotate both reference and polymorphic SVA copies. By characterizing the landscape of both reference and polymorphic SVA retrotransposons, our study enables more accurate genotyping of these elements and facilitate the discovery of pathogenic SVA insertions.

A landscape of complex tandem repeats within individual human genomes.

Markedly expanded tandem repeats (TRs) have been correlated with ~60 diseases. TR diversity has been considered a clue toward understanding missing heritability. However, haplotype-resolved long TRs remain mostly hidden or blacked out because their complex structures (TRs composed of various units and minisatellites containing >10-bp units) make them difficult to determine accurately with existing methods. Here, using a high-precision algorithm to determine complex TR structures from long, accurate reads of PacBio HiFi, an investigation of 270 Japanese control samples yields several genome-wide findings. Approximately 322,000 TRs are difficult to impute from the surrounding single-nucleotide variants. Greater genetic divergence of TR loci is significantly correlated with more events of younger replication slippage. Complex TRs are more abundant than single-unit TRs, and a tendency for complex TRs to consist of <10-bp units and single-unit TRs to be minisatellites is statistically significant at loci with ≥500-bp TRs. Of note, 8909 loci with extended TRs (>100b longer than the mode) contain several known disease-associated TRs and are considered candidates for association with disorders. Overall, complex TRs and minisatellites are found to be abundant and diverse, even in genetically small Japanese populations, yielding insights into the landscape of long TRs.

Investigation of virulence factors, phylogenetic grouping, multiple-locus variable number tandem repeat analysis, and antimicrobial susceptibility of E. coli isolated from aborted bovine fetal tissue.

Escherichia coli is an important microorganism for cattle breeding. The aim of this study was to investigate the presence of phylogenetic groups, virulence factors, genotyping with multi-locus variable tandem repeat analysis (MLVA), and susceptibility to commonly used antimicrobial agents in E. coli strains isolated from aborted bovine fetal samples. In this study, phylogrouping and various virulence genes were analyzed by PCR in E. coli strains isolated from 637 bovine fetal tissue samples. Consequently, E. coli was isolated and identified in 24 samples in culture. Of the 24 isolates identified as positive, 12.5% were defined as group A, 83.3% as B1, and 4.2% as group B2. Of the E. coli isolates, virulence factor fimH was identified in eight (33.3%), traT in 15 (62.5%), ompT in five (20.8%), CNF1 in one (4.16%), and CNF2 in six (25%). Seven genotypic groups were determined as a result of the analysis with the MLVA 10 method. According to the antimicrobial susceptibility test results, high resistance was determined against amoxicillin/clavulanic acid and oxytetracycline. In conclusion, strains of E. coli containing CNF1, CNF2, fimH, traT, and ompT virulence factors can be associated with bovine abortions. It is noteworthy that the dominant phylogenetic group B1 has been observed in cases of cattle abortions.