Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction

A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments.</p

Rare and low-frequency coding variants alter human adult height

Height is a highly heritable, classic polygenic trait with ~700 common associated variants identified so far through genome - wide association studies . Here , we report 83 height - associated coding variants with lower minor allele frequenc ies ( range of 0.1 - 4.8% ) and effects of up to 2 16 cm /allele ( e.g. in IHH , STC2 , AR and CRISPLD2 ) , >10 times the average effect of common variants . In functional follow - up studies, rare height - increasing alleles of STC2 (+1 - 2 cm/allele) compromise d proteolytic inhibition of PAPP - A and increased cleavage of IGFBP - 4 in vitro , resulting in higher bioavailability of insulin - like growth factors . The se 83 height - associated variants overlap genes mutated in monogenic growth disorders and highlight new biological candidates ( e.g. ADAMTS3, IL11RA, NOX4 ) and pathways ( e.g . proteoglycan/ glycosaminoglycan synthesis ) involved in growth . Our results demonstrate that sufficiently large sample sizes can uncover rare and low - frequency variants of moderate to large effect associated with polygenic human phenotypes , and that these variants implicate relevant genes and pathways

Twist exome capture allows for lower average sequence coverage in clinical exome sequencing

Background Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques. Results We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection. Conclusion We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques

Functional genomics and candidate genes for meat quality traits in pigs

El cerdo es la especie doméstica más importante económicamente, no solo por ser la principal fuente de carne en la dieta humana, sino también por su papel como modelo animal para la investigación biomédica. La buena compresión de los mecanismos moleculares que afectan a la calidad de la carne en cerdos es esencial para la obtención de carnes con un patrón saludable de ácidos grasos sin afectar el rendimiento productivo. La composición de ácidos grasos es un carácter muy importante para la calidad de la carne; no obstante, nuestro conocimiento sobre la compleja red de procesos y vías metabólicas que forman el metabolismo de los ácidos grasos sigue siendo limitado. Esta tesis tiene como objetivo general mejorar el entendimiento de estos procesos moleculares, con el fin de comprender mejor la arquitectura genética de la calidad de la carne en el cerdo. Se ha evaluado la implicación funcional de la variante DQ144454:c.2645G<A en la expresión del ACSL4, observando en hígado una mayor expresión en los animales con el alelo G en comparación con los que presentan el alelo A. Se realizó un estudio de asociación (eGWAS) con la expresión génica de ACSL4 identificando dos regiones en los cromosomas 6 y 12 con genes que pueden estar relacionados con la expresión del gen. El gen ELOVL6 ha sido analizado como gen candidato para el QTL del cromosoma 8, relacionado con el contenido de ácidos palmítico y palmitoleico. Se ha determinado la arquitectura genética del gen ELOVL6, mostrando dos isoformas expresadas en hígado y tejido adiposo. El SNP ELOVL6:c.-533C>T presentó una alta asociación con la expresión del propio gen y el contenido de los ácidos palmítico y palmitoleico en músculo y tejido adiposo. Estudios funcionales del promotor del gen validaron la unión de SREBF1 y ERα, sugiriendo un papel importante de ambos factores de transcripción en la regulación del gen ELOVL6. Curiosamente, el SNP ELOVL6:c.-394G>A se encuentra en desequilibrio de ligamiento con el SNP ELOVL6:c.-533C>T y también se encuentra localizado en el único lugar de unión de ERα en el promotor. Además, los estudios realizados permitieron observar una unión diferencial de ERα en función del genotipo del ELOVL6:c.-394G>A, mostrando su unión solo en los animales portadores del alelo G. La unión de ERα ha sido asociada al reclutamiento de metilasas y, consecuentemente, al aumento de los niveles de metilación de los motivos CpG adyacentes. Por lo tanto, las diferencias en la unión de ERα y en el grado de metilación del promotor pueden ser los factores causales de la menor expresión del gen en animales con el alelo G. Por este motivo proponemos el SNP ELOVL6:c.-394G>A como el principal SNP causal de las variaciones fenotípicas del QTL. Finalmente, el transcriptoma del tejido adiposo de cerdos fenotípicamente extremos para la composición de ácidos grasos en músculo fue secuenciado mediante RNA-Seq. Se realizó un análisis de expresión diferencial que permitió identificar 396 genes diferencialmente expresados que modulaban principalmente la lipogénesis, probablemente debido a las diferencias en el contenido de ácidos grasos poliinsaturados entre grupos. Este efecto de la composición de ácidos grasos sobre la expresión de genes lipídicos sugiere que los genes diferencialmente expresados pueden desempeñar un papel importante en el metabolismo de los lípidos y de los ácidos grasos.Pig is one of the most economically important domestic species, since they have been extensively used not only as the major human meat source, but also for biomedical research. A good understanding of the molecular mechanisms affecting meat quality traits in pigs is essential for obtaining meat with a fatty acids profile more in line with public health recommendations without affecting the production yield. Food fatty acid composition is highly relevant for determining meat quality traits, but its metabolism is composed by a complex network of processes and pathways that are not fully understood. Elucidating these molecular processes is the general objective of this thesis, in order to better understand the genetic architecture of meat quality traits in pigs. We evaluated the functional implication of the genetic variant DQ144454:c.2645G<A on ACSL4 gene expression, observing in liver a higher expression in animals with the G allele than animals with the A allele. A SNP genome-wide association study with ACSL4 gene expression was performed and two significant regions on SSC6 and SSC12 with candidate genes associated with ACSL4 gene expression were identified. On the other hand, ELOVL6 gene was analyzed as a candidate gene for the QTL on SSC8 affecting palmitic and palmitoleic acid content. The complete genetic architecture of ELOVL6 gene was described, showing two different isoforms in liver and adipose tissue. The ELOVL6:c.-533C>T SNP, identified in the promoter region, was highly associated with its own gene expression and the percentages of palmitic and palmitoleic fatty acids in muscle and adipose tissue. Functional analyses of ELOVL6 promoter showed the occupancy of SREBF1 and ERα, suggesting an important role of both transcription factors on the regulation of ELOVL6 gene expression. Interestingly, the ELOVL6:c.-394G>A SNP is in linkage disequilibrium with ELOVL6:c.-533C>T SNP and also is located within the only predicted ERE on ELOVL6 promoter. In addition, ChIP assays showed that ERα binding depends on ELOVL6:c.-394G>A genotype, showing union only in animals carrying the G allele. It has been described that ERα binding causes the recruitment of Dnmts and, consequently, an increase on the methylation levels of the surrounding CpG motifs. Therefore, differences on ERα binding and the methylation pattern may be the causal factors of the lower ELOVL6 expression in animals with the ELOVL6:c.-394G allele. Hence, we proposed ELOVL6:c.-394G>A SNP as the major putative causal mutation for explaining the phenotypic variation of the QTL analyzed. Finally, the adipose tissue transcriptomes of two groups of phenotypically extreme pigs for intramuscular fatty acid composition were sequenced using RNA-Seq. Differential expression analysis identified 396 genes differentially expressed between groups. The major metabolic pathway differentially modulated between groups was lipogenesis, probably caused by the differences on PUFA content between the two groups. Therefore, the linked effect of fatty acid composition on lipid-related gene expression suggested that differentially-expressed genes may play an important role in lipid and fatty acid metabolism

Functional genomics and candidate genes for meat quality traits in pigs

El cerdo es la especie doméstica más importante económicamente, no solo por ser la principal fuente de carne en la dieta humana, sino también por su papel como modelo animal para la investigación biomédica. La buena compresión de los mecanismos moleculares que afectan a la calidad de la carne en cerdos es esencial para la obtención de carnes con un patrón saludable de ácidos grasos sin afectar el rendimiento productivo. La composición de ácidos grasos es un carácter muy importante para la calidad de la carne; no obstante, nuestro conocimiento sobre la compleja red de procesos y vías metabólicas que forman el metabolismo de los ácidos grasos sigue siendo limitado. Esta tesis tiene como objetivo general mejorar el entendimiento de estos procesos moleculares, con el fin de comprender mejor la arquitectura genética de la calidad de la carne en el cerdo. Se ha evaluado la implicación funcional de la variante DQ144454:c.2645GT presentó una alta asociación con la expresión del propio gen y el contenido de los ácidos palmítico y palmitoleico en músculo y tejido adiposo. Estudios funcionales del promotor del gen validaron la unión de SREBF1 y ERα, sugiriendo un papel importante de ambos factores de transcripción en la regulación del gen ELOVL6. Curiosamente, el SNP ELOVL6:c.-394G>A se encuentra en desequilibrio de ligamiento con el SNP ELOVL6:c.-533C>T y también se encuentra localizado en el único lugar de unión de ERα en el promotor. Además, los estudios realizados permitieron observar una unión diferencial de ERα en función del genotipo del ELOVL6:c.-394G>A, mostrando su unión solo en los animales portadores del alelo G. La unión de ERα ha sido asociada al reclutamiento de metilasas y, consecuentemente, al aumento de los niveles de metilación de los motivos CpG adyacentes. Por lo tanto, las diferencias en la unión de ERα y en el grado de metilación del promotor pueden ser los factores causales de la menor expresión del gen en animales con el alelo G. Por este motivo proponemos el SNP ELOVL6:c.-394G>A como el principal SNP causal de las variaciones fenotípicas del QTL. Finalmente, el transcriptoma del tejido adiposo de cerdos fenotípicamente extremos para la composición de ácidos grasos en músculo fue secuenciado mediante RNA-Seq. Se realizó un análisis de expresión diferencial que permitió identificar 396 genes diferencialmente expresados que modulaban principalmente la lipogénesis, probablemente debido a las diferencias en el contenido de ácidos grasos poliinsaturados entre grupos. Este efecto de la composición de ácidos grasos sobre la expresión de genes lipídicos sugiere que los genes diferencialmente expresados pueden desempeñar un papel importante en el metabolismo de los lípidos y de los ácidos grasos.Pig is one of the most economically important domestic species, since they have been extensively used not only as the major human meat source, but also for biomedical research. A good understanding of the molecular mechanisms affecting meat quality traits in pigs is essential for obtaining meat with a fatty acids profile more in line with public health recommendations without affecting the production yield. Food fatty acid composition is highly relevant for determining meat quality traits, but its metabolism is composed by a complex network of processes and pathways that are not fully understood. Elucidating these molecular processes is the general objective of this thesis, in order to better understand the genetic architecture of meat quality traits in pigs. We evaluated the functional implication of the genetic variant DQ144454:c.2645GT SNP, identified in the promoter region, was highly associated with its own gene expression and the percentages of palmitic and palmitoleic fatty acids in muscle and adipose tissue. Functional analyses of ELOVL6 promoter showed the occupancy of SREBF1 and ERα, suggesting an important role of both transcription factors on the regulation of ELOVL6 gene expression. Interestingly, the ELOVL6:c.-394G>A SNP is in linkage disequilibrium with ELOVL6:c.-533C>T SNP and also is located within the only predicted ERE on ELOVL6 promoter. In addition, ChIP assays showed that ERα binding depends on ELOVL6:c.-394G>A genotype, showing union only in animals carrying the G allele. It has been described that ERα binding causes the recruitment of Dnmts and, consequently, an increase on the methylation levels of the surrounding CpG motifs. Therefore, differences on ERα binding and the methylation pattern may be the causal factors of the lower ELOVL6 expression in animals with the ELOVL6:c.-394G allele. Hence, we proposed ELOVL6:c.-394G>A SNP as the major putative causal mutation for explaining the phenotypic variation of the QTL analyzed. Finally, the adipose tissue transcriptomes of two groups of phenotypically extreme pigs for intramuscular fatty acid composition were sequenced using RNA-Seq. Differential expression analysis identified 396 genes differentially expressed between groups. The major metabolic pathway differentially modulated between groups was lipogenesis, probably caused by the differences on PUFA content between the two groups. Therefore, the linked effect of fatty acid composition on lipid-related gene expression suggested that differentially-expressed genes may play an important role in lipid and fatty acid metabolism

Common and rare variants in patients with early onset drusen maculopathy

Early onset drusen maculopathy (EODM) can lead to advanced macular degeneration at a young age, affecting quality of life. However, the genetic causes of EODM are not well studied. We performed whole genome sequencing in 49 EODM patients. Common genetic variants were analysed by calculating genetic risk scores based on 52 age-related macular generation (AMD)-associated variants, and we analysed rare variants in candidate genes to identify potential deleterious variants that might contribute to EODM development. We demonstrate that the 52 AMD-associated variants contributed to EODM, especially variants located in the complement pathway. Furthermore, we identified 26 rare genetic variants predicted to be pathogenic based on in silico prediction tools or based on reported pathogenicity in literature. These variants are located predominantly in the complement and lipid metabolism pathways. Last, evaluation of 18 genes causing inherited retinal dystrophies that can mimic AMD characteristics, revealed 11 potential deleterious variants in eight EODM patients. However, phenotypic characteristics did not point towards a retinal dystrophy in these patients. In conclusion, this study reports new insights into rare variants that are potentially involved in EODM development, and which are relevant for future studies unravelling the aetiology of EODM

Integration of liver gene co-expression networks and eGWAs analyses highlighted candidate regulators implicated in lipid metabolism in pigs

Altres ajuts: M. Revilla was funded by a Formació i Contractació de Personal Investigador Novell (FI-DGR) PhD grant from Generalitat de Catalunya(ECO/1639/2013).In the present study, liver co-expression networks and expression Genome Wide Association Study (eGWAS) were performed to identify DNA variants and molecular pathways implicated in the functional regulatory mechanisms of meat quality traits in pigs. With this purpose, the liver mRNA expression of 44 candidates genes related with lipid metabolism was analysed in 111 Iberian x Landrace backcross animals. The eGWAS identified 92 eSNPs located in seven chromosomal regions and associated with eight genes: CROT, CYP2U1, DGAT1, EGF, FABP1, FABP5, PLA2G12A, and PPARA. Remarkably, cis-eSNPs associated with FABP1 gene expression which may be determining the C18:2(n-6)/C18:3(n-3) ratio in backfat through the multiple interaction of DNA variants and genes were identified. Furthermore, a hotspot on SSC8 associated with the gene expression of eight genes was identified and the TBCK gene was pointed out as candidate gene regulating it. Our results also suggested that the PI3K-Akt-mTOR pathway plays an important role in the control of the analysed genes highlighting nuclear receptors as the NR3C1 or PPARA. Finally, sex-dimorphism associated with hepatic lipid metabolism was identified with over-representation of female-biased genes. These results increase our knowledge of the genetic architecture underlying fat composition traits

Twist exome capture allows for lower average sequence coverage in clinical exome sequencing

Abstract Background Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques. Results We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection. Conclusion We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques

Twist exome capture allows for lower average sequence coverage in clinical exome sequencing.

BACKGROUND: Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques. RESULTS: We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection. CONCLUSION: We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques

Liver transcriptome profile in pigs with extreme phenotypes of intramuscular fatty acid composition

Background: New advances in high-throughput technologies have allowed for the massive analysis of genomic data, providing new opportunities for the characterization of the transcriptome architectures. Recent studies in pigs have employed RNA-Seq to explore the transcriptome of different tissues in a reduced number of animals. The main goal of this study was the identification of differentially-expressed genes in the liver of Iberian x Landrace crossbred pigs showing extreme phenotypes for intramuscular fatty acid composition using RNA-Seq. Results: The liver transcriptomes of two female groups (H and L) with phenotypically extreme intramuscular fatty acid composition were sequenced using RNA-Seq. A total of 146 and 180 unannotated protein-coding genes were identified in intergenic regions for the L and H groups, respectively. In addition, a range of 5.8 to 7.3% of repetitive elements was found, with SINEs being the most abundant elements. The expression in liver of 186 (L) and 270 (H) lncRNAs was also detected. The higher reproducibility of the RNA-Seq data was validated by RT-qPCR and porcine expression microarrays, therefore showing a strong correlation between RT-qPCR and RNA-Seq data (ranking from 0.79 to 0.96), as well as between microarrays and RNA-Seq (r=0.72). A differential expression analysis between H and L animals identified 55 genes differentially-expressed between groups. Pathways analysis revealed that these genes belong to biological functions, canonical pathways and three gene networks related to lipid and fatty acid metabolism. In concordance with the phenotypic classification, the pathways analysis inferred that linolenic and arachidonic acids metabolism was altered between extreme individuals. In addition, a connection was observed among the top three networks, hence suggesting that these genes are interconnected and play an important role in lipid and fatty acid metabolism. Conclusions: In the present study RNA-Seq was used as a tool to explore the liver transcriptome of pigs with extreme phenotypes for intramuscular fatty acid composition. The differential gene expression analysis showed potential gene networks which affect lipid and fatty acid metabolism. These results may help in the design of selection strategies to improve the sensorial and nutritional quality of pork meat