Luces y sombras del análisis de expresión génica utilizando microarrays. Un ejemplo en cerdo ibérico

Ponencia publicada en ITEA, vol.104La tecnología de los microarrays de expresión es la herramienta ideal para el estudio de patrones de expresión de miles de genes de forma simultánea. Sin embargo existe gran variabilidad de resultados atribuible a los aspectos técnicos y de análisis estadístico. En este trabajo presentamos algunos de los problemas surgidos en el estudio de las diferencias de expresión en hígado de cerdos ibéricos para los tratamientos sexo y alimentación empleando microarrays de Affymetrix. Los datos de expresión normalizados fueron analizados siguiendo dos aproximaciones de la metodología de los modelos mixtos. Para ambos tratamientos las diferencias de expresión detectadas fueron dependientes del modelo de análisis y solo un pequeño número de genes diferencialmente expresados fueron coincidentes en ambas estrategias estadísticas. Algunas de estas diferencias de expresión fueron validadas por PCR cuantitativa. Además identificamos errores de diseño y falta de anotación de las sondas del array. Los resultados de este estudio nos han permitido detectar diferencias de expresión de algunos genes de interés, pero también remarcan la necesidad de realizar estudios complementarios que confirmen las diferencias de expresión reveladas a través de la tecnología de los microarraysLights and darkness of gene expression analysis using microarrays: an example in Iberian pigs Expression microarray technology is the ideal tool for the study of thousands of gene expression patterns simultaneously. However there is a great variability of results attributed to technical and statistical analysis aspects. In this work we present several of the arisen problems of a differential expression study in liver of Iberian pigs under the treatments sex and feeding level using Affymetrix microarray. Normalized expression data were analyzed following two approaches of the mixed model methodology. In both treatments the detected differential expressions were dependent of the statistical model and just a small number of genes were coincident between both statistical strategies. Some of the expression differences were confirmed by quantitative PCR. Besides, we have identified design mistakes and missing annotation of the array probes. The results of this study have allowed us to detect differential expression of interesting genes, but it pointed out the necessity of carrying out complementary studies in order to confirm the differential expressions revealed using microarrays technology

Diversidad genética de las razas porcinas ibérica y alentejana mediante el genotipado de variantes genéticas en genes mayores y genes candidatos

Las razas porcinas ibérica y alentejana se encuentran biogeográficamente localizadas en el suroeste de la península Ibérica. Estas razas comparten un conjunto de características comunes. Ambas son fenotípicamente muy similares; muestran un bajo índice de crecimiento y una elevada deposición grasa y tienen un sistema común de producción al aire libre en el que se explotan los recursos de la Dehesa. El objetivo del presente trabajo, dentro del marco del proyecto TREASURE, consistió en caracterizar la diversidad genética de ambas razas usando los datos de genotipado de 39 polimorfismos localizados en 34 genes previamente asociados a distintos caracteres de interés económico. Estos polimorfismos fueron genotipados en 950 cerdos pertenecientes a las razas ibérica y alentejana y a 18 razas europeas autóctonas adicionales. Los bajos valores para heterozigosidad observada y esperada indican una baja diversidad genética en ibéricos y alentejanos. Las distancias genéticas estimadas usando DS y FST fueron cercanas a 0 (0.007 y 0.039, respectivamente), sugieren que estas razas son genéticamente muy similares. Asimismo, las técnicas de análisis multivariante utilizadas como análisis de componentes principales y asignación a grupos mostraron que los individuos de estas razas están muy próximos formando clusters diferenciados del resto de razas. Estos resultados concuerdan con los de otros autores que ya mostraron distancias genéticas cortas entre ambas razas usando secuencias de ADN mitocondrial

Comparative RNAseq analysis of backfat tissue from local pig breeds.

Alentejano (AL) and Bísaro (BI) are the main local pig breeds in Portugal, but have no information comparing their transcriptomic activity. AL belongs to the Iberian branch, presenting lower growth rates, precociously high adipogenic activity and higher levels of unsaturated fatty acids (FAs) while BI pig is from the Celtic group, sharing ancestors with higher growth rate and leaner commercial breeds. This work intended to explore the genome function of AL and BI to better understand the underlying physiological mechanisms associated with body fat accretion, lipid composition and meat quality. Dorsal subcutaneous fat (DSF) samples were collected from AL and BI fattening pigs, with ~150kg BW at slaughter. Total RNA was obtained and sequenced for transcriptome analysis. Bioinformatic analyses using three different tools (Cufflinks, EdgeR and DESeq2) were performed. A total of 367, 137 and 155 differentially expressed genes (DEGs) (q-value0.8) were found using the Cufflinks, EdgeR and DESeq2 pipelines, respectively, between AL and BI DSF samples. EdgeR and DESeq2 shared a total 121 DEGs (~71% overlap) while Cufflinks showed divergent results (2.7% overlap with EdgeR and 5.5% with DESeq2). A functional enrichment analysis of the candidate DEGs was performed using Ingenuity Pathway Analysis. Synthesis of lipid, depletion of glycogen, mass of organism and accumulation of oleic acid were revealed as main involved functions (p-value<0.05) though no directional activation state was observed (-2<Zscore<2). Potential upstream regulators that explain the obtained results such as TCF7L2 and RIPK2 were predicted to be activated and inhibited in AL, respectively. Moreover, 4 causal networks with RIT2, KL, FLCN and RIPK2 as master regulators were inhibited in AL while another with PPARGC1B was activated. These results present the first high-throughput transcriptomic data involving these local breeds and can help explain the metabolic differences that occur in the adipose tissue and shed light into specific meat quality traits

Taking advantage from phenotype variability in a local animal genetic resource: identification of genomic regions associated with the hairless phenotype in Casertana pigs

Casertana is an endangered autochthonous pig breed (raised in south-central Italy) that is considered to be the descendant of the influential Neapolitan pig population that was used to improve British breeds in the 19th century. Casertana pigs are characterized by a typical, almost complete, hairless phenotype, even though a few Casertana pigs are normal haired. In this work, using Illumina PorcineSNP60 BeadChip data, we carried out a genome-wide association study and an FST analysis with this breed by comparing animals showing the classical hairless phenotype (n = 81) versus pigs classified as haired (n = 15). Combining the results obtained with the two approaches, we identified two significant regions: one on porcine chromosome (SSC) 7 and one on SSC15. The SSC7 region contains the forkhead box N3 (FOXN3) gene, the most plausible candidate gene of this region, considering that mutations in another gene of the same family (forkhead box N1; Foxn1 or FOXN1) are responsible for the nude locus in rodents and alopecia in humans. Another potential candidate gene, rho guanine nucleotide exchange factor 10 (ARHGEF10), is located in the SSC15 region. FOXN3 and ARHGEF10 have been detected as differentially expressed in androgenetic and senescent alopecia respectively. This study on an autochthonous pig breed contributes to shed some light on novel genes potentially involved in hair development and growth and demonstrates that local animal breeds can be valuable genetic resources for disclosing genetic factors affecting unique traits, taking advantage of phenotype variability segregating in small populations

Genetic diversity of Alentejano and Iberian breeds assessed by polymorphisms of major genes

Alentejano and Iberian breeds are biogeographically located in the southwest of the Iberian Peninsula. These breeds shared a set of common characteristics. Both are phenotypically very similar showing a low growth rate and a high intramuscular fat content and are reared under extensive systems in open range fields. The aim of the current study, carried out within the framework of the TREASURE project, intends to characterize of the genetic diversity of Alentejano and Iberian breeds using the genotyping data of 32 polymorphisms located on 26 major genes. These polymorphisms were genotyped in a total of 950 animals belonging to Alentejano, Iberian and 18 additional European breeds. The low mean values of observed (Ho) and expected (Hs) heterozygosity and FIS point out a low genetic diversity in the analyzed breeds. The genetic distances estimated using Ds and FST revealed values were close to 0 (0.005 and 0.030, respectively), suggesting that these breeds are genetically similar. The population structure analyzed with multivariate methods such as Discriminant Analysis of Principal Component and admixture analyses showed that both breeds were grouped within the same cluster. These results are supported by other authors based on mitochondrial sequences who reported short genetic distances between these breeds. Although complementary analyses using a larger number of markers should be performed, the results of the current analyses support the hypothesis of Alentejano and Iberian could be different strains of the same breed

Identification of differentially expressed key genes of Longissimus lumborum samples from Portuguese Alentejano and Bísaro local pig breeds

Most of the swine industry nowadays is about productive and profitable fast-growing lean breeds raised under intensive conditions leading to meat and fat rich in saturated fatty acids. The Portuguese local Alentejano (AL) and Bísaro (BI) pig breeds present high intramuscular fat (IMF) content which contributes to highly appreciated pork products. These breeds have different ancestors: AL belongs to the Iberian group, presenting lower growth rates and higher lipid accretion and unsaturated fatty acids level when compared to BI, which belongs to the Celtic group, sharing ancestors with leaner breeds such as Large White and Landrace. The goal of this work was to compare the muscle gene expression profiles of AL and BI pig breeds to better understand the influence of the genetic background in the main metabolic processes occurring in the Longissimus lumborum (LL) muscle, namely in terms of lipid synthesis, muscle tissue formation, protein synthesis and cell proliferation. LL samples were obtained at slaughter, from adult AL and BI pigs with ~150kg body weight. Total RNA was extracted and sequenced for a transcriptome comparison analysis. A total of 250 genes were found to be differentially expressed (DE) in LL samples (q<0.05) conditional on breed, with 174 DE genes up-regulated in AL (log2(fold_change) = 0.65 to 7.03) and 76 in BI (log2(fold_change) = 0.63 to 4.53). Genes related to skeletal muscle development and function, such as MYH3, MYH13 or ACTN4, were significantly up-regulated in BI when compared to AL, which is in agreement with the higher muscle mass of the former breed. Genes involved in lipid metabolism were up-regulated in AL, including SCD (q=0.05), responsible for catalysing the reaction that introduces the first double bond into saturated fatty acyl-CoA substrates, which agrees with the higher unsaturation of fat tissues generally associated with the former breed. A functional enrichment analysis (metabolic pathways and GO enrichment) was performed for the DE genes and the identified functions included tissue development, cellular growth and proliferation, quantity of connective tissue and lipid metabolism. Potential regulators found that explain the observed gene expression changes in the dataset included molecules such as: ADORA2A, CEBPA, SMAD3 and PPARG (predicted to be activated in AL); HDAC and ASXL1 (predicted to be inhibited in AL)

Comparative Transcriptomic Analysis of Subcutaneous Adipose Tissue from Local Pig Breeds

When compared to modern lean-type breeds, Portuguese local Alentejano (AL) and Bísaro (BI) pig breeds present a high potential for subcutaneous and intramuscular fat (IMF) deposition which contributes for better meat quality. The aim of this work was to explore the genome function to better understand the underlying physiological mechanisms associated with body fat accretion. Dorsal subcutaneous fat samples were collected at slaughter from adult animals (n = 4 for each breed) with ~150 kg body weight. Total RNA was obtained and sequenced for transcriptome analysis using DESeq2. A total of 458 di erentially expressed (DE) genes (q-value < 0.05) were identified, with 263 overexpressed in AL and 195 in BI. Key genes involved in de novo fatty acid biosynthesis, elongation and desaturation were upregulated in AL such as ACLY, FASN, ME1, ELOVL6 and SCD. A functional enrichment analysis of the DE genes was performed using Ingenuity Pathway Analysis. Cholesterol synthesis is suggested to be higher in AL via SREBF2, SCAP and PPARG, while lipolytic activity may be more active in BI through GH and AMPK signalling. Increased signalling of CD40 together with the predicted activation of INSIG1 and INSIG2 in BI suggests that this breed is more sensitive to insulin whereas the AL is less sensitive like the Iberian breed

Transcriptomic profiling of skeletal muscle reveals candidate genes influencing muscle growth and associated lipid composition in Portuguese local pig breeds

Gene expression is one of the main factors to influence meat quality by modulating fatty acid metabolism, composition, and deposition rates in muscle tissue. This study aimed to explore the transcriptomics of the Longissimus lumborum muscle in two local pig breeds with distinct genetic background using next-generation sequencing technology and Real-Time qPCR. RNA-seq yielded 49 differentially expressed genes between breeds, 34 overexpressed in the Alentejano (AL) and 15 in the Bísaro (BI) breed. Specific slow type myosin heavy chain components were associated with AL (MYH7) and BI (MYH3) pigs, while an overexpression of MAP3K14 in AL may be associated with their lower loin proportion, induced insulin resistance, and increased inflammatory response via NFkB activation. Overexpression of RUFY1 in AL pigs may explain the higher intramuscular (IMF) content via higher GLUT4 recruitment and consequently higher glucose uptake that can be stored as fat. Several candidate genes for lipid metabolism, excluded in the RNA-seq analysis due to low counts, such as ACLY, ADIPOQ, ELOVL6, LEP and ME1 were identified by qPCR as main gene factors defining the processes that influence meat composition and quality. These results agree with the fatter profile of the AL pig breed and adiponectin resistance can be postulated as responsible for the overexpression of MAP3K140s coding product NIK, failing to restore insulin sensitivity

Influencia de la composición de la dieta sobre el perfil de ácidos grasos y la expresión génica en tejidos adiposo, muscular y hepático de cerdos ibéricos

La composición de los tejidos animales es determinante en la calidad de los productos y está influida por varios factores como la dieta, el tipo genético, la edad y el sexo. En este trabajo se ha evaluado el efecto de la composición de ácidos grasos (AG) de la dieta de cerdos ibéricos en fase de cebo, sobre la composición de AG de los tejidos y la transcripción de genes codificantes para enzimas clave del metabolismo lipídico (SCD, ME1, FASN, ACACA, LEP, CPT, HADH). Se utilizaron 40 machos Torbiscal que recibieron diferentes dietas: saturada (S), monoinsaturada (M) y poliinsaturada (P). La composición de AG de los tejidos adiposo, hepático y muscular mostró grandes diferencias del grupo P respecto a M y S, que mostraron un perfil similar. La dieta afectó también a la expresión génica en hígado y tejido adiposo, sugiriendo una mayor expresión de enzimas lipogénicas en el grupo M y menor en el P. Estos resultados no explican la mayor capacidad del grupo S para la síntesis endógena de AG, que podría deducirse de los análisis de composición tisular.La composición de los tejidos animales es determinante en la calidad de los productos y está influida por varios factores como la dieta, el tipo genético, la edad y el sexo. En este trabajo se ha evaluado el efecto de la composición de ácidos grasos (AG) de la dieta de cerdos ibéricos en fase de cebo, sobre la composición de AG de los tejidos y la transcripción de genes codificantes para enzimas clave del metabolismo lipídico (SCD, ME1, FASN, ACACA, LEP, CPT, HADH). Se utilizaron 40 machos Torbiscal que recibieron diferentes dietas: saturada (S), monoinsaturada (M) y poliinsaturada (P). La composición de AG de los tejidos adiposo, hepático y muscular mostró grandes diferencias del grupo P respecto a M y S, que mostraron un perfil similar. La dieta afectó también a la expresión génica en hígado y tejido adiposo, sugiriendo una mayor expresión de enzimas lipogénicas en el grupo M y menor en el P. Estos resultados no explican la mayor capacidad del grupo S para la síntesis endógena de AG, que podría deducirse de los análisis de composición tisular

Structural differences among pig genomes illustrate genetic uniqueness of breeds

The availability of high-throughput whole-genome sequencing (WGS) data illustrating differences among different pig breed genomes opened a new area of genomic research focused on variation caused by single nucleotide polymorphisms (SNP), small scale variation and structural variants which may all contribute to phenotypic variation among pig breeds. In our study (performed within TREASURE project) we re-analysed WGS-based data sets from more than 20 breeds, including commercial and local breeds as well as some wild boar genomes, deposited in publicly available databases. This bioinformatics tool enables discovery of new SNPs, estimation of allele frequencies (genotyping by sequencing) at candidate loci and identification of structural variation in a wide range of pig breeds. The analysis underlined the relevance of structural differences at KIT and MC1R locus involved in colour pattern formation, as well as LEPR locus associated with fatness, fatty acid metabolism and intramuscular fat composition. This approach allows discovery of important genomic differences between commercial breeds and local breeds which are analysed in the frame of the TREASURE project. Extensive mining of publicly available genomic data can together with the newly generated genomic information from local breeds, significantly contribute to the detailed characterisation of animal genetic resources present in local pig breeds. Funded by European Union’s H2020 RIA program (grant agreement No. 634476)