HIGH- RESOLUTION MELTING (HRM) CURVE ANALYSIS: NEW APPROACH USED TO DETECT BLAD AND DUMPS IN URUGUAYAN HOLSTEIN BREED

The widespread use of artificial insemination has allowed the expansion of genetic progress. However, it also brought consequences such as the expansion of lethal hereditary diseases and the increase in inbreeding. The object of this study was to establish a fast and sensitive molecular assay to detect bovine leukocyte adhesion deficiency (BLAD) and deficiency of uridine monophosphate synthase (DUMPS) carriers in Uruguayan Holstein cattle by means of high resolution melting (HRM) curve analysis. By testing previously confirmed carrier and non-carrier animals, we set up a rapid, simple, and inexpensive diagnostic test using PCR followed by HRM curve analysis. The PCR-HRM genotyping method was effective for the discrimination of BLAD and DUMPS homozygous genotypes, and the BLAD heterozygous genotype. We conclude that the PCR-HRM assay is a robust, reliable, and economical tool for the detection of these mutations in the Holstein breed, which may be implemented in genetic selection programs

IDENTIFICATION OF HOLSTEIN COWS CARRIERS OF COMPLEX VERTEBRAL MALFORMATION BY HIGH RESOLUTION MELTING CURVES (HRM)

The objective of this study was the optimization and implementation of a reliable and economical molecular screening method for the detection of the mutant allele of CVM (complex vertebral malformation, c.559G>T, SLC35A3) by HRM analysis, as well as analyzing its existence in a representative sample of Holstein cows from the Milk Genomic DNA Bank of Uruguay. The optimization of the HRM methodology in the RotorGene™ 6000 equipment (Corbett Life Science, Australia) by amplification of the 79 bp PCR products clearly differentiated the two genotypes: homozygous, wild type: GG; and heterozygous, carrier for the mutation CVM: GT (c.559G>T; SLC35A3). In the analyzed sample, the frequency of the mutant allele (T) for CVM was high (q = 0.032), with a prevalence of carrier cows of 6.45%. It is concluded that the PCR-HRM analysis is a fast, easily interpretable, low cost, and highly accurate technique for the detection of this mutation in Holstein cattle, which may be implemented in genetic selection programs

Familial hypercholesterolaemia in children and adolescents from 48 countries: a cross-sectional study

Background: Approximately 450 000 children are born with familial hypercholesterolaemia worldwide every year, yet only 2·1% of adults with familial hypercholesterolaemia were diagnosed before age 18 years via current diagnostic approaches, which are derived from observations in adults. We aimed to characterise children and adolescents with heterozygous familial hypercholesterolaemia (HeFH) and understand current approaches to the identification and management of familial hypercholesterolaemia to inform future public health strategies. Methods: For this cross-sectional study, we assessed children and adolescents younger than 18 years with a clinical or genetic diagnosis of HeFH at the time of entry into the Familial Hypercholesterolaemia Studies Collaboration (FHSC) registry between Oct 1, 2015, and Jan 31, 2021. Data in the registry were collected from 55 regional or national registries in 48 countries. Diagnoses relying on self-reported history of familial hypercholesterolaemia and suspected secondary hypercholesterolaemia were excluded from the registry; people with untreated LDL cholesterol (LDL-C) of at least 13·0 mmol/L were excluded from this study. Data were assessed overall and by WHO region, World Bank country income status, age, diagnostic criteria, and index-case status. The main outcome of this study was to assess current identification and management of children and adolescents with familial hypercholesterolaemia. Findings: Of 63 093 individuals in the FHSC registry, 11 848 (18·8%) were children or adolescents younger than 18 years with HeFH and were included in this study; 5756 (50·2%) of 11 476 included individuals were female and 5720 (49·8%) were male. Sex data were missing for 372 (3·1%) of 11 848 individuals. Median age at registry entry was 9·6 years (IQR 5·8-13·2). 10 099 (89·9%) of 11 235 included individuals had a final genetically confirmed diagnosis of familial hypercholesterolaemia and 1136 (10·1%) had a clinical diagnosis. Genetically confirmed diagnosis data or clinical diagnosis data were missing for 613 (5·2%) of 11 848 individuals. Genetic diagnosis was more common in children and adolescents from high-income countries (9427 [92·4%] of 10 202) than in children and adolescents from non-high-income countries (199 [48·0%] of 415). 3414 (31·6%) of 10 804 children or adolescents were index cases. Familial-hypercholesterolaemia-related physical signs, cardiovascular risk factors, and cardiovascular disease were uncommon, but were more common in non-high-income countries. 7557 (72·4%) of 10 428 included children or adolescents were not taking lipid-lowering medication (LLM) and had a median LDL-C of 5·00 mmol/L (IQR 4·05-6·08). Compared with genetic diagnosis, the use of unadapted clinical criteria intended for use in adults and reliant on more extreme phenotypes could result in 50-75% of children and adolescents with familial hypercholesterolaemia not being identified. Interpretation: Clinical characteristics observed in adults with familial hypercholesterolaemia are uncommon in children and adolescents with familial hypercholesterolaemia, hence detection in this age group relies on measurement of LDL-C and genetic confirmation. Where genetic testing is unavailable, increased availability and use of LDL-C measurements in the first few years of life could help reduce the current gap between prevalence and detection, enabling increased use of combination LLM to reach recommended LDL-C targets early in life

Análise da expressão gênica global da bactéria Xylella fastidiosa em laranja doce por microarranjos de DNA

Foi construído um microarranjo com as 2600 ORFs identificadas no projeto de sequenciamento da bactéria Xylella fastidiosa estirpe 9a5c, e utilizado para analisar diferenças na expressão gênica global da bactéria dentro de uma laranja doce suscetível (Pera) e uma tolerante (cultivar Navelina ISA 315). Foram achados mais genes diferencialmente expressos envolvidos na degradação, reguladores, componentes de membrana, adesinas tipo fímbrias, transportadores, elementos genéticos móveis e genes de patogenicidade na variedade sintomática. Assim, na cultivar Navelina ISA 315, foram diferencialmente expressos mais genes relacionados com a resposta ao estresse, seja detoxificação de espécies reativas do oxigênio ou proteínas chaperonas, assim como uma adesina do tipo hemaglutinina. Isso sugere diferenças na agregação celular e composição do biofilme assim como um maior estresse da bactéria na cultivar tolerante, provocado pelas próprias defesas da planta ou por microrganismos endofíticos que estão competindo com a X. fastidiosa. A técnica de microarranjos foi validada pela RT-qPCR, e apresentou-se como uma ferramenta poderosa na análise das mudanças na expressão gênica da bactéria X. fastidiosa em plantas de laranja doce in vivo, apresentando uma visão mais real da natureza do que os sistemas in vitro, que não a conseguem imitar completamente. Foram levantadas neste trabalho algumas hipóteses sobre os mecanismos de patogenicidade mas no entanto, mais pesquisas ainda são necessárias para lograr melhor compreensão dos mecanismos de patogenicidade e das interações patógeno-hospedeiroA DNA microarray was constructed containing 2600 ORFs identified by the Genome sequencing project of Xylella fastidiosa 9a5c strain, and used to check global gene expression differences in the bacteria within a susceptible and a tolerant sweet orange plant, the variety Pera and the cultivar Navelina ISA 315, respectively. More genes related to degradation, regulation, membrane components, fimbrial adhesins, transport, genetic mobile elements and patogenicity genes were differentially expressed in Pera variety. On the other hand, in the cultivar Navelina ISA 315, more genes related to stress response, detoxification of oxygen reactive species or other substances, “heat shock” proteins, as well as an adhesin of the hemagglutinin type, suggesting differences in cellular aggregation and biofilm composition, as well as a higher estress of the bacteria in tolerant cultivar, produced either by plant defenses or by endophitic microorganisms which are competing with X. fastidiosa. This “handmade” DNA microchip was validated by RT-qPCR, and has revealed as a powerful technique for the analysis of global changes in gene expression of X. fastidiosa in sweet orange plants in vivo, generating a more real image of what is happening in nature than in vitro systems which would never reproduce nature conditions exactly. Some hypotheses were raised in this study about patogenicity mechanisms, therefore, more research is still necessary to achieve a better understanding of pathogenicity mechanisms and host-pathogen interactionsCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

RESEARCH ARTICLE - Analysis of genetic diversity in the Oryza officinalis complex

The genetic relationships among 34 accessions of wild rice from Asia, Africa, America and Australia were analysed using RFLP technique. After southern blotting, DNA digestion pattern was hybridised with a highly repetitive DNA sequence of a retrotransposon from a gypsy family of mobile elements. A dendrogram was constructed from RFLP data in which the species clustered according to their genome designation (CC, BB, BBCC and CCDD genomes). Some species did not appear in the same group, for example, O. eichingeri from Africa and Sri Lanka clustered separately from each other. The same situation was observed for the accessions from China of O. officinalis, which cluster together showing a close relationship with O. rhizomatis, and O. eichingeri (both of CC genome). Also, the tetraploid BBCC from India of O. officinalis appears in the same cluster of O. eichingeri and O. punctata (both from Africa) suggesting close phylogenetic relationship with the African genomes BB, CC and BBCC

Analysis of Uruguayan weedy rice genetic diversity using AFLP molecular markers

Weedy rice is a serious problem in Uruguayan rice fields since intensification of rice production started about 10 years ago. The genetic diversity of 26 weedy accessions of weedy rice and 6 Uruguayan cultivars were analyzed using AFLP (amplified fragment length polymorphisms) methodology. Abundant polymorphisms were found among samples tested. Using different methods of analysis three groups of samples were revealed. A relationship was found between three groups and morphological traits. One group had a black hull, purple apex and long awn (wild type traits) while another group had straw hull and apex, and short or no awn (domestication traits). The third group included the cultivars analyzed and some weedy rice samples. The weedy rice in this third group is presumed to most closely mimic cultivated rice and may have recently evolved. The results suggest that weedy rice adapts either to the natural environment or to cultivation. The former type with black hull and long awn may be easy to control because it can easily be seen. The later group may be difficult to control, particularly since the weedy rices within the cluster consisting of cultivars suggest that weedy rices are continually evolving in Uruguayan rice fields. The AFLP technique is very effective for assessing genetic diversity within weedy rice and will be very useful for fingerprinting of local cultivars of rice

Proceedings Of The 23Rd Paediatric Rheumatology European Society Congress: Part Two

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