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Functional Impact and Evolution of a Novel Human Polymorphic Inversion That Disrupts a Gene and Creates a Fusion Transcript.

Puig M, Castellano D, Pantano L, Giner-Delgado C, Izquierdo D, Gayà-Vidal M, Lucas-Lledó JI, Esko T, Terao C, Matsuda F, Cáceres M - PLoS Genet. (2015)

Bottom Line: PCR genotyping in 541 individuals from eight different human populations allowed the detection of tag SNPs and inversion genotyping in multiple populations worldwide, showing that the inverted allele is mainly found in East Asia with an average frequency of 4.7%.Interestingly, one of the breakpoints disrupts the transcription factor gene ZNF257, causing a significant reduction in the total expression level of this gene in lymphoblastoid cell lines.Moreover, we have found a new fusion transcript that is generated exclusively from inverted chromosomes around one of the breakpoints.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain.

ABSTRACT
Despite many years of study into inversions, very little is known about their functional consequences, especially in humans. A common hypothesis is that the selective value of inversions stems in part from their effects on nearby genes, although evidence of this in natural populations is almost nonexistent. Here we present a global analysis of a new 415-kb polymorphic inversion that is among the longest ones found in humans and is the first with clear position effects. This inversion is located in chromosome 19 and has been generated by non-homologous end joining between blocks of transposable elements with low identity. PCR genotyping in 541 individuals from eight different human populations allowed the detection of tag SNPs and inversion genotyping in multiple populations worldwide, showing that the inverted allele is mainly found in East Asia with an average frequency of 4.7%. Interestingly, one of the breakpoints disrupts the transcription factor gene ZNF257, causing a significant reduction in the total expression level of this gene in lymphoblastoid cell lines. RNA-Seq analysis of the effects of this expression change in standard homozygotes and inversion heterozygotes revealed distinct expression patterns that were validated by quantitative RT-PCR. Moreover, we have found a new fusion transcript that is generated exclusively from inverted chromosomes around one of the breakpoints. Finally, by the analysis of the associated nucleotide variation, we have estimated that the inversion was generated ~40,000-50,000 years ago and, while a neutral evolution cannot be ruled out, its current frequencies are more consistent with those expected for a deleterious variant, although no significant association with phenotypic traits has been found so far.

No MeSH data available.


Related in: MedlinePlus

Analysis of the new fusion transcript in HsInv0379 breakpoint.A. RNA-Seq reads mapped to an AC construct corresponding to BP1 (vertical red arrow) in an inverted chromosome reveal a fusion transcript present only in the four Std/Inv individuals (bottom) but not in the four Std/Std (top). Boxes highlight the new exon (yellow) and ZNF257 first exon (green). The structure of the fusion transcript reconstructed by Cufflinks [38] is shown below the RNA-Seq profiles. Small arrows indicate the approximate position of the primers used to validate the transcript. The coordinates of the new exon in the HG19 reference genome are also indicated. B. Analysis of fusion transcript expression by RT-PCR in several individuals. C. Quantification of the fusion transcript levels by qPCR in 15 Std/Std, 11 Std/Inv, and 1 Inv/Inv individuals.
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pgen.1005495.g004: Analysis of the new fusion transcript in HsInv0379 breakpoint.A. RNA-Seq reads mapped to an AC construct corresponding to BP1 (vertical red arrow) in an inverted chromosome reveal a fusion transcript present only in the four Std/Inv individuals (bottom) but not in the four Std/Std (top). Boxes highlight the new exon (yellow) and ZNF257 first exon (green). The structure of the fusion transcript reconstructed by Cufflinks [38] is shown below the RNA-Seq profiles. Small arrows indicate the approximate position of the primers used to validate the transcript. The coordinates of the new exon in the HG19 reference genome are also indicated. B. Analysis of fusion transcript expression by RT-PCR in several individuals. C. Quantification of the fusion transcript levels by qPCR in 15 Std/Std, 11 Std/Inv, and 1 Inv/Inv individuals.

Mentions: Another important effect of the inversion is that it brings the ZNF257 promoter and first exons to a completely different part of the genome, where they might induce the transcription of new chimeric transcripts. Therefore, we specifically searched for transcripts generated by the ZNF257 promoter around BP1 in Inv chromosomes. By mapping RNA-Seq reads to a construct with the inverted sequence, we detected a spliced fusion transcript formed by the first exon of gene ZNF257 and a completely new 296-bp exon made up of fragments of LINE and Alu elements (Fig 4). We confirmed the existence of this RNA by amplifying most of it by RT-PCR and by sequencing the exon-exon junction in individual NA18956. Indeed, both by RT-PCR and qPCR the fusion transcript was found in the 11 inversion carriers analyzed, with 1.7-fold higher expression levels in the Inv/Inv individual, while it is not expressed in any of the Std/Std individuals tested (including three non-East Asian individuals) (Fig 4 and S2 Fig). The complete new transcript is short, with only 468 bases, and its longest ORF contains only 75 aa. It does not show homology to any other known transcript either. However, according to the RNA-Seq reads mapping to the ZNF257 exon 1 (common to the two genes) in inversion heterozygotes that express both transcripts and in Std homozygotes (Fig 4), the level of expression of the fusion transcript is at least 7.4 times higher than that of ZNF257.


Functional Impact and Evolution of a Novel Human Polymorphic Inversion That Disrupts a Gene and Creates a Fusion Transcript.

Puig M, Castellano D, Pantano L, Giner-Delgado C, Izquierdo D, Gayà-Vidal M, Lucas-Lledó JI, Esko T, Terao C, Matsuda F, Cáceres M - PLoS Genet. (2015)

Analysis of the new fusion transcript in HsInv0379 breakpoint.A. RNA-Seq reads mapped to an AC construct corresponding to BP1 (vertical red arrow) in an inverted chromosome reveal a fusion transcript present only in the four Std/Inv individuals (bottom) but not in the four Std/Std (top). Boxes highlight the new exon (yellow) and ZNF257 first exon (green). The structure of the fusion transcript reconstructed by Cufflinks [38] is shown below the RNA-Seq profiles. Small arrows indicate the approximate position of the primers used to validate the transcript. The coordinates of the new exon in the HG19 reference genome are also indicated. B. Analysis of fusion transcript expression by RT-PCR in several individuals. C. Quantification of the fusion transcript levels by qPCR in 15 Std/Std, 11 Std/Inv, and 1 Inv/Inv individuals.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591017&req=5

pgen.1005495.g004: Analysis of the new fusion transcript in HsInv0379 breakpoint.A. RNA-Seq reads mapped to an AC construct corresponding to BP1 (vertical red arrow) in an inverted chromosome reveal a fusion transcript present only in the four Std/Inv individuals (bottom) but not in the four Std/Std (top). Boxes highlight the new exon (yellow) and ZNF257 first exon (green). The structure of the fusion transcript reconstructed by Cufflinks [38] is shown below the RNA-Seq profiles. Small arrows indicate the approximate position of the primers used to validate the transcript. The coordinates of the new exon in the HG19 reference genome are also indicated. B. Analysis of fusion transcript expression by RT-PCR in several individuals. C. Quantification of the fusion transcript levels by qPCR in 15 Std/Std, 11 Std/Inv, and 1 Inv/Inv individuals.
Mentions: Another important effect of the inversion is that it brings the ZNF257 promoter and first exons to a completely different part of the genome, where they might induce the transcription of new chimeric transcripts. Therefore, we specifically searched for transcripts generated by the ZNF257 promoter around BP1 in Inv chromosomes. By mapping RNA-Seq reads to a construct with the inverted sequence, we detected a spliced fusion transcript formed by the first exon of gene ZNF257 and a completely new 296-bp exon made up of fragments of LINE and Alu elements (Fig 4). We confirmed the existence of this RNA by amplifying most of it by RT-PCR and by sequencing the exon-exon junction in individual NA18956. Indeed, both by RT-PCR and qPCR the fusion transcript was found in the 11 inversion carriers analyzed, with 1.7-fold higher expression levels in the Inv/Inv individual, while it is not expressed in any of the Std/Std individuals tested (including three non-East Asian individuals) (Fig 4 and S2 Fig). The complete new transcript is short, with only 468 bases, and its longest ORF contains only 75 aa. It does not show homology to any other known transcript either. However, according to the RNA-Seq reads mapping to the ZNF257 exon 1 (common to the two genes) in inversion heterozygotes that express both transcripts and in Std homozygotes (Fig 4), the level of expression of the fusion transcript is at least 7.4 times higher than that of ZNF257.

Bottom Line: PCR genotyping in 541 individuals from eight different human populations allowed the detection of tag SNPs and inversion genotyping in multiple populations worldwide, showing that the inverted allele is mainly found in East Asia with an average frequency of 4.7%.Interestingly, one of the breakpoints disrupts the transcription factor gene ZNF257, causing a significant reduction in the total expression level of this gene in lymphoblastoid cell lines.Moreover, we have found a new fusion transcript that is generated exclusively from inverted chromosomes around one of the breakpoints.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain.

ABSTRACT
Despite many years of study into inversions, very little is known about their functional consequences, especially in humans. A common hypothesis is that the selective value of inversions stems in part from their effects on nearby genes, although evidence of this in natural populations is almost nonexistent. Here we present a global analysis of a new 415-kb polymorphic inversion that is among the longest ones found in humans and is the first with clear position effects. This inversion is located in chromosome 19 and has been generated by non-homologous end joining between blocks of transposable elements with low identity. PCR genotyping in 541 individuals from eight different human populations allowed the detection of tag SNPs and inversion genotyping in multiple populations worldwide, showing that the inverted allele is mainly found in East Asia with an average frequency of 4.7%. Interestingly, one of the breakpoints disrupts the transcription factor gene ZNF257, causing a significant reduction in the total expression level of this gene in lymphoblastoid cell lines. RNA-Seq analysis of the effects of this expression change in standard homozygotes and inversion heterozygotes revealed distinct expression patterns that were validated by quantitative RT-PCR. Moreover, we have found a new fusion transcript that is generated exclusively from inverted chromosomes around one of the breakpoints. Finally, by the analysis of the associated nucleotide variation, we have estimated that the inversion was generated ~40,000-50,000 years ago and, while a neutral evolution cannot be ruled out, its current frequencies are more consistent with those expected for a deleterious variant, although no significant association with phenotypic traits has been found so far.

No MeSH data available.


Related in: MedlinePlus