Dataset Information

A deletion in the intergenic region upstream of Ednrb causes head spot in the rat strain KFRS4/Kyo.

ABSTRACT: Head spot is one of the phenotypes identified in the KFRS4/Kyo rat strain. Although previous linkage analysis suggested that Ednrb, which is frequently involved in coat color variations in various animals, could be the gene responsible for this phenotype, no mutations have been identified in its coding region.To identify mutations causative of this phenotype in KFRS4/Kyo, we analyzed target capture sequencing data that we recently generated. Our target capture method has a unique feature, i.e., it covers not only exonic regions but also conserved non-coding sequences (CNSs) among vertebrates; therefore, it has the potential to detect regulatory mutations. We identified a deletion of approximately 50 kb in length approximately 50 kb upstream of Ednrb. A comparative analysis with the epigenomic data in the corresponding region in humans and mice showed that one of the CNSs might be an enhancer. Further comparison with Hi-C data, which provide information about chromosome conformation, indicated that the putative enhancer is spatially close to the promoter of Ednrb, suggesting that it acts as an enhancer of Ednrb.These in silico data analyses strongly suggest that the identified deletion in the intergenic region upstream of Ednrb, which might contain a melanocyte-specific enhancer, is the mutation causative of the head spot phenotype in the KFRS4/Kyo rat strain.

SUBMITTER: Yoshihara M

PROVIDER: S-EPMC5372274 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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A deletion in the intergenic region upstream of Ednrb causes head spot in the rat strain KFRS4/Kyo.

Yoshihara Minako M Sato Tetsuya T Saito Daisuke D Ohara Osamu O Kuramoto Takashi T Suyama Mikita M

BMC genetics 20170329 1

<h4>Background</h4>Head spot is one of the phenotypes identified in the KFRS4/Kyo rat strain. Although previous linkage analysis suggested that Ednrb, which is frequently involved in coat color variations in various animals, could be the gene responsible for this phenotype, no mutations have been identified in its coding region.<h4>Results</h4>To identify mutations causative of this phenotype in KFRS4/Kyo, we analyzed target capture sequencing data that we recently generated. Our target capture ...[more]

PMID: 28356074

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Project description:SINE-VNTR-Alu (SVA) retrotransposons are a subclass of transposable elements (TEs) that exist only in primate genomes. TE insertions can be co-opted as cis-regulatory elements (CREs); however, the regulatory potential of SVAs has predominantly been demonstrated using bioinformatic approaches and reporter gene assays. The objective of this study was to demonstrate SVA cis-regulatory activity by CRISPR (clustered regularly interspaced short palindromic repeats) deletion and subsequent measurement of direct effects on local gene expression. We identified a region on chromosome 17 that was enriched with human-specific SVAs. Comparative gene expression analysis at this region revealed co-expression of TRPV1 and TRPV3 in multiple human tissues, which was not observed in mouse, highlighting key regulatory differences between the two species. Furthermore, the intergenic region between TRPV1 and TRPV3 coding sequences contained a human specific SVA insertion located upstream of the TRPV3 promoter and downstream of the 3' end of TRPV1, highlighting this SVA as a candidate to study its potential cis-regulatory activity on both genes. Firstly, we generated SVA reporter gene constructs and demonstrated their transcriptional regulatory activity in HEK293 cells. We then devised a dual-targeting CRISPR strategy to facilitate the deletion of this entire SVA sequence and generated edited HEK293 clonal cell lines containing homozygous and heterozygous SVA deletions. In edited homozygous ∆SVA clones, we observed a significant decrease in both TRPV1 and TRPV3 mRNA expression, compared to unedited HEK293. In addition, we also observed an increase in the variability of mRNA expression levels in heterozygous ∆SVA clones. Overall, in edited HEK293 with SVA deletions, we observed a disruption to the co-expression of TRPV1 and TRPV3. Here we provide an example of a human specific SVA with cis-regulatory activity in situ, supporting the role of SVA retrotransposons as contributors to species-specific gene expression.

Project description:Genetic causes of ocular conditions remain largely unknown. To reveal the molecular basis for a congenital ocular phenotype associated with glaucoma we performed whole-exome sequencing (WES) and whole-genome copy number analyses of patient DNA. WES did not identify a causative variant. Copy number variation analysis identified a deletion of 10p13 in the patient and his unaffected father; the deletion breakpoint contained a single 37-bp sequence that is normally present in two distinct Alu repeats separated by ~181 kb. The deletion removed part of the upstream region of optineurin (OPTN) as well as the upstream sequence and two coding exons of coiled-coil domain containing 3 (CCDC3); analysis of the patient's second allele showed normal OPTN and CCDC3 sequences. Studies of zebrafish orthologs identified expression in the developing eye for both genes. OPTN is a known factor in dominant adult-onset glaucoma and Amyotrophic Lateral Sclerosis (ALS). The deletion eliminates 98 kb of the OPTN upstream sequence leaving only ~1 kb of the proximal promoter region. Comparison of transcriptional activation capability of the 3 kb normal and the rearranged del(10)(p13) OPTN promoter sequences demonstrated a statistically significant decrease for the deleted allele; sequence analysis of the entire deleted region identified multiple conserved elements with possible cis-regulatory activity. Additional screening of CCDC3 indicated that heterozygous loss-of-function alleles are unlikely to cause congenital ocular disease. In summary, we report the first regulatory region deletion involving OPTN, caused by Alu-mediated nonallelic homologous recombination and possibly contributing to the patient's ocular phenotype. In addition, our data indicate that Alu-mediated rearrangements of the OPTN upstream region may represent a new source of affected alleles in human conditions. Evaluation of the upstream OPTN sequences in additional ocular and ALS patients may help to determine the role of this region, if any, in human disease.

Dataset Information

A deletion in the intergenic region upstream of Ednrb causes head spot in the rat strain KFRS4/Kyo.

Publications

A deletion in the intergenic region upstream of Ednrb causes head spot in the rat strain KFRS4/Kyo.

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