SummaryAn important requirement for a state‐of‐the‐art hepatitis B surface antigen (HBsAg) screen... more SummaryAn important requirement for a state‐of‐the‐art hepatitis B surface antigen (HBsAg) screening assay is reliable detection of mutated HBsAg. Currently, there is a striking shortage of data regarding the detection rates of in vivo HBsAg mutations for these clinically important assays. Therefore, we compared the detection rates of four commercial HBsAg screening assays using a global cohort of 1553 patients from four continents with known HBV genotypes. These samples, which represent the broadest spectrum of known and novel HBsAg major hydrophilic region (MHR) mutations to date, were analyzed for the presence of HBsAg using the Roche Elecsys® HBsAg II Qualitative, Siemens ADVIA Centaur XP HBsAg II, Abbott Architect HBsAg Qualitative II and DiaSorin Liaison® HBsAg Qualitative assays, respectively. Of the 1553 samples, 1391 samples could be sequenced; of these, 1013 (72.8%) carried at least one of the 345 currently known amino acid substitutions (distinct HBsAg mutation) in the HB...
Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology, Jan 5, 2018
To avoid false negative results, hepatitis B surface antigen (HBsAg) assays need to detect sample... more To avoid false negative results, hepatitis B surface antigen (HBsAg) assays need to detect samples with mutations in the immunodominant 'a' determinant region, which vary by ethnographic region. We evaluated the prevalence and type of HBsAg mutations in a hepatitis B virus (HBV)-infected East- and Southeast Asian population, and the diagnostic performance of the Elecsys HBsAg II Qualitative assay. We analyzed 898 samples from patients with HBV infection from four sites (China [Beijing and Guangzhou], Korea and Vietnam). HBsAg mutations were detected and sequenced using highly sensitive ultra-deep sequencing and compared between the first (amino acids 124-137) and second (amino acids 139-147) loops of the 'a' determinant region using the Elecsys HBsAg II Qualitative assay. Overall, 237 distinct amino acid mutations in the major hydrophilic region were identified; mutations were present in 660 of 898 HBV-infected patient samples (73.5%). Within the pool of 237 distinct...
The diversity of the hepatitis B surface antigen (HBsAg) has a significant impact on the performa... more The diversity of the hepatitis B surface antigen (HBsAg) has a significant impact on the performance of diagnostic screening tests and the clinical outcome of hepatitis B infection. Neutralizing or diagnostic antibodies against the HBsAg are directed towards its highly conserved major hydrophilic region (MHR), in particular towards its "a" determinant subdomain. Here, we explored, on a global scale, the genetic diversity of the HBsAg MHR in a large, multi-ethnic cohort of randomly selected subjects with HBV infection from four continents. A total of 1553 HBsAg positive blood samples of subjects originating from 20 different countries across Africa, America, Asia and central Europe were characterized for amino acid variation in the MHR. Using highly sensitive ultra-deep sequencing, we found 72.8% of the successfully sequenced subjects (n = 1391) demonstrated amino acid sequence variation in the HBsAg MHR. This indicates that the global variation frequency in the HBsAg MHR i...
Legumes are the third largest family of angiosperms and the second most important crop class. Leg... more Legumes are the third largest family of angiosperms and the second most important crop class. Legume genomes have been shaped by extensive large-scale gene duplications, including an approximately 58 million year old whole genome duplication shared by most crop legumes. We report the genome and the transcription atlas of coding and non-coding genes of a Mesoamerican genotype of common bean (Phaseolus vulgaris L., BAT93). Using a comprehensive phylogenomics analysis, we assessed the past and recent evolution of common bean, and traced the diversification of patterns of gene expression following duplication. We find that successive rounds of gene duplications in legumes have shaped tissue and developmental expression, leading to increased levels of specialization in larger gene families. We also find that many long non-coding RNAs are preferentially expressed in germ-line-related tissues (pods and seeds), suggesting that they play a significant role in fruit development. Our results also suggest that most bean-specific gene family expansions, including resistance gene clusters, predate the split of the Mesoamerican and Andean gene pools. The genome and transcriptome data herein generated for a Mesoamerican genotype represent a counterpart to the genomic resources already available for the Andean gene pool. Altogether, this information will allow the genetic dissection of the characters involved in the domestication and adaptation of the crop, and their further implementation in breeding strategies for this important crop.
Mitochondrial disorders are a frequent cause of neurological disability affecting children and ad... more Mitochondrial disorders are a frequent cause of neurological disability affecting children and adults. Traditionally, molecular diagnosis of mitochondrial diseases was mostly accomplished by the use of Sanger sequencing and PCR-RFLP. However, there are particular drawbacks associated with the use of these methods. Recent multidisciplinary advances have led to new sequencing methods that may overcome these limitations. Our goal was to explore the use of a next generation sequencing platform in the molecular diagnosis of mitochondrial diseases reporting our findings in adult patients that present with a clinical-pathological diagnosis of a mitochondrial encephalomyopathy. Complete genomic sequences of mitochondrial DNA were obtained by 454 massive pyrosequencing from blood samples. The analysis of these sequences allowed us to identify two diagnostic pathogenic mutations and 74 homoplasmic polymorphisms, useful for obtaining high-resolution mitochondrial haplogroups. In summary, molecular diagnosis of mitochondrial disorders could be efficiently done from readily accessible samples, such as blood, with the use of a new sequencing platform.
Chronic coinfection with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) is among ... more Chronic coinfection with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) is among the greatest challenges facing public health worldwide. In this population, the response to hepatitis C therapy by treatment with pegylated interferon plus ribavirin (PEG-IFN+RBV) is lower than in HCV-monoinfected patients, particularly in those infected by HCV genotype 1. A PKR/eIF-2α phosphorylation homology domain (PePHD) within the E2 protein has been found to interact with PKR and inhibit PKR in vitro, suggesting a possible mechanism for HCV to evade the antiviral effects of IFN. The aim of this work was to analyze the amino acid conservation in the HCV-E2-PePHD and quasispecies diversity among HCV-HIV-coinfected patients exhibiting sustained virological response, non-response, or partial response with viral relapse to PEG-IFN+RBV by ultra-deep pyrosequencing. For this purpose, HCV-E2-PePHD PCR products were generated and sequenced directly for four patients with a sustained response, seven patients with no virological response, and four patients with viral relapse before and after treatment with PEG-IFN+RBV. HCV-E2-PePHD amino acid sequences were obtained for isolates from serum collected before and during treatment (24 h, 4 weeks, and 12 weeks). Quasispecies analysis of the HCV-E2-PePHD and flanking genomic regions was performed using 454/Roche pyrosequencing, analyzing 39,364 sequence reads in total. The HCV-E2-PePHD sequence at the amino acid and nucleotide level was highly conserved among HCV genotype 1 strains, irrespective of the PEG-IFN+RBV response. This high degree of amino acid conservation and sporadic mutations in the HCV-E2-PePHD domain do not appear to be associated with treatment outcome. The HCV-E2-PePHD sequence before or during treatment cannot be used to predict reliably the outcome of treatment in patients coinfected with HCV genotype 1 and HIV.
ABSTRACT The TcZFPs are a family of small zinc finger proteins harboring WW domains or Proline ri... more ABSTRACT The TcZFPs are a family of small zinc finger proteins harboring WW domains or Proline rich motifs. In Trypanosoma brucei, ZFPs are involved during stage specific differentiation. TcZFPs interact with each other using the WW domain (ZFP2 and ZFP3) and the proline rich motif (ZFP1). The tcZFP1b member is exclusive to Trypanosoma cruzi and it is only expressed in trypomastigote stage. We used a tetracycline inducible vector to express ectopically tcZFP1b in the epimastigote stage. Upon induction of tcZFP1b, the parasites stopped dividing completely after five days. Visual inspection showed abnormal distorted-morphology (monster) cells with multiple flagella and increased DNA contents. We were interested in investigate global transcription changes occurred during the generation of this abnormal phenotype. Thus, we performed RNA-seq transcriptome profiling with a 454 pyrosequencer to analyze the global changes after ectopic expression of tcZFP1b. The total mRNAs sequenced from induced and non-induced control epimastigotes showed, after filtering the data, a set of 70 genes having equal or more than 3X fold change upregulation, while 35 genes showed equal or more than 3X fold downregulation. Interestingly, several trans-sialidase-like genes and pseudogenes were upregulated along with several genes in the categories of amino acid catabolism and carbohydrate metabolism. On the other hand, hypothetical proteins, fatty acid biosynthesis and mitochondrial functions dominated the group of downregulated genes. Our data showed that several mRNAs sharing related functions and pathways changed their levels in a concerted pattern resembling post-transcriptional regulons. We also found two different motifs in the 3'UTRs of the majority of mRNAs, one for upregulated and other for downregulated genes
Mitochondrial disorders are a frequent cause of neurological disability affecting children and ad... more Mitochondrial disorders are a frequent cause of neurological disability affecting children and adults. Traditionally, molecular diagnosis of mitochondrial diseases was mostly accomplished by the use of Sanger sequencing and PCR-RFLP. However, there are particular drawbacks associated with the use of these methods. Recent multidisciplinary advances have led to new sequencing methods that may overcome these limitations. Our goal was to explore the use of a next generation sequencing platform in the molecular diagnosis of mitochondrial diseases reporting our findings in adult patients that present with a clinical-pathological diagnosis of a mitochondrial encephalomyopathy. Complete genomic sequences of mitochondrial DNA were obtained by 454 massive pyrosequencing from blood samples. The analysis of these sequences allowed us to identify two diagnostic pathogenic mutations and 74 homoplasmic polymorphisms, useful for obtaining high-resolution mitochondrial haplogroups. In summary, molecular diagnosis of mitochondrial disorders could be efficiently done from readily accessible samples, such as blood, with the use of a new sequencing platform.
SummaryAn important requirement for a state‐of‐the‐art hepatitis B surface antigen (HBsAg) screen... more SummaryAn important requirement for a state‐of‐the‐art hepatitis B surface antigen (HBsAg) screening assay is reliable detection of mutated HBsAg. Currently, there is a striking shortage of data regarding the detection rates of in vivo HBsAg mutations for these clinically important assays. Therefore, we compared the detection rates of four commercial HBsAg screening assays using a global cohort of 1553 patients from four continents with known HBV genotypes. These samples, which represent the broadest spectrum of known and novel HBsAg major hydrophilic region (MHR) mutations to date, were analyzed for the presence of HBsAg using the Roche Elecsys® HBsAg II Qualitative, Siemens ADVIA Centaur XP HBsAg II, Abbott Architect HBsAg Qualitative II and DiaSorin Liaison® HBsAg Qualitative assays, respectively. Of the 1553 samples, 1391 samples could be sequenced; of these, 1013 (72.8%) carried at least one of the 345 currently known amino acid substitutions (distinct HBsAg mutation) in the HB...
Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology, Jan 5, 2018
To avoid false negative results, hepatitis B surface antigen (HBsAg) assays need to detect sample... more To avoid false negative results, hepatitis B surface antigen (HBsAg) assays need to detect samples with mutations in the immunodominant 'a' determinant region, which vary by ethnographic region. We evaluated the prevalence and type of HBsAg mutations in a hepatitis B virus (HBV)-infected East- and Southeast Asian population, and the diagnostic performance of the Elecsys HBsAg II Qualitative assay. We analyzed 898 samples from patients with HBV infection from four sites (China [Beijing and Guangzhou], Korea and Vietnam). HBsAg mutations were detected and sequenced using highly sensitive ultra-deep sequencing and compared between the first (amino acids 124-137) and second (amino acids 139-147) loops of the 'a' determinant region using the Elecsys HBsAg II Qualitative assay. Overall, 237 distinct amino acid mutations in the major hydrophilic region were identified; mutations were present in 660 of 898 HBV-infected patient samples (73.5%). Within the pool of 237 distinct...
The diversity of the hepatitis B surface antigen (HBsAg) has a significant impact on the performa... more The diversity of the hepatitis B surface antigen (HBsAg) has a significant impact on the performance of diagnostic screening tests and the clinical outcome of hepatitis B infection. Neutralizing or diagnostic antibodies against the HBsAg are directed towards its highly conserved major hydrophilic region (MHR), in particular towards its "a" determinant subdomain. Here, we explored, on a global scale, the genetic diversity of the HBsAg MHR in a large, multi-ethnic cohort of randomly selected subjects with HBV infection from four continents. A total of 1553 HBsAg positive blood samples of subjects originating from 20 different countries across Africa, America, Asia and central Europe were characterized for amino acid variation in the MHR. Using highly sensitive ultra-deep sequencing, we found 72.8% of the successfully sequenced subjects (n = 1391) demonstrated amino acid sequence variation in the HBsAg MHR. This indicates that the global variation frequency in the HBsAg MHR i...
Legumes are the third largest family of angiosperms and the second most important crop class. Leg... more Legumes are the third largest family of angiosperms and the second most important crop class. Legume genomes have been shaped by extensive large-scale gene duplications, including an approximately 58 million year old whole genome duplication shared by most crop legumes. We report the genome and the transcription atlas of coding and non-coding genes of a Mesoamerican genotype of common bean (Phaseolus vulgaris L., BAT93). Using a comprehensive phylogenomics analysis, we assessed the past and recent evolution of common bean, and traced the diversification of patterns of gene expression following duplication. We find that successive rounds of gene duplications in legumes have shaped tissue and developmental expression, leading to increased levels of specialization in larger gene families. We also find that many long non-coding RNAs are preferentially expressed in germ-line-related tissues (pods and seeds), suggesting that they play a significant role in fruit development. Our results also suggest that most bean-specific gene family expansions, including resistance gene clusters, predate the split of the Mesoamerican and Andean gene pools. The genome and transcriptome data herein generated for a Mesoamerican genotype represent a counterpart to the genomic resources already available for the Andean gene pool. Altogether, this information will allow the genetic dissection of the characters involved in the domestication and adaptation of the crop, and their further implementation in breeding strategies for this important crop.
Mitochondrial disorders are a frequent cause of neurological disability affecting children and ad... more Mitochondrial disorders are a frequent cause of neurological disability affecting children and adults. Traditionally, molecular diagnosis of mitochondrial diseases was mostly accomplished by the use of Sanger sequencing and PCR-RFLP. However, there are particular drawbacks associated with the use of these methods. Recent multidisciplinary advances have led to new sequencing methods that may overcome these limitations. Our goal was to explore the use of a next generation sequencing platform in the molecular diagnosis of mitochondrial diseases reporting our findings in adult patients that present with a clinical-pathological diagnosis of a mitochondrial encephalomyopathy. Complete genomic sequences of mitochondrial DNA were obtained by 454 massive pyrosequencing from blood samples. The analysis of these sequences allowed us to identify two diagnostic pathogenic mutations and 74 homoplasmic polymorphisms, useful for obtaining high-resolution mitochondrial haplogroups. In summary, molecular diagnosis of mitochondrial disorders could be efficiently done from readily accessible samples, such as blood, with the use of a new sequencing platform.
Chronic coinfection with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) is among ... more Chronic coinfection with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) is among the greatest challenges facing public health worldwide. In this population, the response to hepatitis C therapy by treatment with pegylated interferon plus ribavirin (PEG-IFN+RBV) is lower than in HCV-monoinfected patients, particularly in those infected by HCV genotype 1. A PKR/eIF-2α phosphorylation homology domain (PePHD) within the E2 protein has been found to interact with PKR and inhibit PKR in vitro, suggesting a possible mechanism for HCV to evade the antiviral effects of IFN. The aim of this work was to analyze the amino acid conservation in the HCV-E2-PePHD and quasispecies diversity among HCV-HIV-coinfected patients exhibiting sustained virological response, non-response, or partial response with viral relapse to PEG-IFN+RBV by ultra-deep pyrosequencing. For this purpose, HCV-E2-PePHD PCR products were generated and sequenced directly for four patients with a sustained response, seven patients with no virological response, and four patients with viral relapse before and after treatment with PEG-IFN+RBV. HCV-E2-PePHD amino acid sequences were obtained for isolates from serum collected before and during treatment (24 h, 4 weeks, and 12 weeks). Quasispecies analysis of the HCV-E2-PePHD and flanking genomic regions was performed using 454/Roche pyrosequencing, analyzing 39,364 sequence reads in total. The HCV-E2-PePHD sequence at the amino acid and nucleotide level was highly conserved among HCV genotype 1 strains, irrespective of the PEG-IFN+RBV response. This high degree of amino acid conservation and sporadic mutations in the HCV-E2-PePHD domain do not appear to be associated with treatment outcome. The HCV-E2-PePHD sequence before or during treatment cannot be used to predict reliably the outcome of treatment in patients coinfected with HCV genotype 1 and HIV.
ABSTRACT The TcZFPs are a family of small zinc finger proteins harboring WW domains or Proline ri... more ABSTRACT The TcZFPs are a family of small zinc finger proteins harboring WW domains or Proline rich motifs. In Trypanosoma brucei, ZFPs are involved during stage specific differentiation. TcZFPs interact with each other using the WW domain (ZFP2 and ZFP3) and the proline rich motif (ZFP1). The tcZFP1b member is exclusive to Trypanosoma cruzi and it is only expressed in trypomastigote stage. We used a tetracycline inducible vector to express ectopically tcZFP1b in the epimastigote stage. Upon induction of tcZFP1b, the parasites stopped dividing completely after five days. Visual inspection showed abnormal distorted-morphology (monster) cells with multiple flagella and increased DNA contents. We were interested in investigate global transcription changes occurred during the generation of this abnormal phenotype. Thus, we performed RNA-seq transcriptome profiling with a 454 pyrosequencer to analyze the global changes after ectopic expression of tcZFP1b. The total mRNAs sequenced from induced and non-induced control epimastigotes showed, after filtering the data, a set of 70 genes having equal or more than 3X fold change upregulation, while 35 genes showed equal or more than 3X fold downregulation. Interestingly, several trans-sialidase-like genes and pseudogenes were upregulated along with several genes in the categories of amino acid catabolism and carbohydrate metabolism. On the other hand, hypothetical proteins, fatty acid biosynthesis and mitochondrial functions dominated the group of downregulated genes. Our data showed that several mRNAs sharing related functions and pathways changed their levels in a concerted pattern resembling post-transcriptional regulons. We also found two different motifs in the 3'UTRs of the majority of mRNAs, one for upregulated and other for downregulated genes
Mitochondrial disorders are a frequent cause of neurological disability affecting children and ad... more Mitochondrial disorders are a frequent cause of neurological disability affecting children and adults. Traditionally, molecular diagnosis of mitochondrial diseases was mostly accomplished by the use of Sanger sequencing and PCR-RFLP. However, there are particular drawbacks associated with the use of these methods. Recent multidisciplinary advances have led to new sequencing methods that may overcome these limitations. Our goal was to explore the use of a next generation sequencing platform in the molecular diagnosis of mitochondrial diseases reporting our findings in adult patients that present with a clinical-pathological diagnosis of a mitochondrial encephalomyopathy. Complete genomic sequences of mitochondrial DNA were obtained by 454 massive pyrosequencing from blood samples. The analysis of these sequences allowed us to identify two diagnostic pathogenic mutations and 74 homoplasmic polymorphisms, useful for obtaining high-resolution mitochondrial haplogroups. In summary, molecular diagnosis of mitochondrial disorders could be efficiently done from readily accessible samples, such as blood, with the use of a new sequencing platform.
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Papers by Gaston Westergaard