A deletion in the intergenic region upstream of Ednrb causes head spot in the rat strain KFRS4/Kyo
© The Author(s). 2017
Received: 11 January 2017
Accepted: 25 March 2017
Published: 29 March 2017
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.
KeywordsTarget capture sequencing Exome Conserved non-coding sequence Regulatory mutation Comparative genomics Rat Head spot
Fancy rat colonies carry various mutations, such as those for coat color and pattern . These are among the most prominent phenotypes often observed in other domesticated animals, such as dogs, cats, horses, and mice. KFRS4/Kyo is an inbred rat strain derived from the crossing of a fancy rat with the PVG/Seac rat strain, which possesses various phenotypes, including white spotting on the head (head spot; hs) and abnormal outer ear morphology (dumbo; dmbo), both of which were shown to be autosomal recessive phenotypes . Although a mutation causative of the dumbo phenotype was successfully identified by fine-scale linkage analysis  followed by genome resequencing , the mutation causative of the head spot phenotype still remains to be identified . To date, a linkage analysis has been performed on the head spot phenotype, and the mutation responsible for it was shown to reside in chr15:84.6–91.2 Mb (rat rn4 assembly) .
Several genes involved in coat color variations have been identified . Ednrb is one such gene responsible for distinctive coat color phenotypes in various species, for example, Waardenburg syndrome type 4 (WS4) in humans , aganglionosis in rats [5–7], piebald-lethal in mice , Lethal White Foal Syndrome in horses , and panda plumage color phenotype in Japanese quails , some of these species possess a characteristic head spot. In the case of the head spot phenotype in KFRS4/Kyo, Ednrb was suspected to be the gene responsible for this phenotype because it is located within the genomic interval identified by the linkage analysis. However, no mutations were found in the gene body of Ednrb in KFRS4/Kyo . One possible explanation for these results is the existence of a mutation associated with Ednrb gene regulation somewhere around Ednrb in the interval identified by the linkage analysis.
There are several approaches to identify a gene responsible for a certain phenotype. These include linkage analysis, genome-wide association study, and exome sequencing. Among these methods, exome sequencing has the potential to directly identify a mutation causative of a phenotype, and has been widely used in the field of genomics of diseases since its first application to identify a mutation responsible for a genetic disorder . Although exome sequencing has been successfully applied to identify mutations causative of many genetic disorders (for a review, see ), it has a drawback in that it can only identify mutations in the target regions, namely, annotated exons and the neighboring sequences, and it cannot, in principle, detect causative mutations if they are located in deep intergenic or intronic regions. To partly overcome this drawback while maintaining efficiency in terms of not sequencing genomic regions with lower selective constraints, we recently devised a novel target capture sequencing method, TargetEC, which can detect mutations not only in exonic parts of genes but also in conserved non-coding sequences that are thought to be important for gene regulation .
In this study, we analyzed our data obtained by the TargetEC method  to identify a mutation causative of head spot in KFRS4/Kyo rats, and found a candidate deletion in the intergenic region upstream of Ednrb. We utilized the data from a linkage analysis, as well as in silico comparative analyses of epigenomic data obtained from international consortia [14, 15] and chromosome conformation data obtained from Hi-C analysis of human and mouse cell lines [16, 17], to obtain evidence supportive of our hypothesis that this deletion might contain a melanocyte-specific enhancer of Ednrb.
We used the sequence read data of the KFRS4/Kyo inbred strain generated by the TargetEC method, which is a target capture method focusing only on exons and conserved non-coding sequences (CNSs), obtained from our previous study (DDBJ Sequence Read Archive accession number: DRA004543) . In conventional exome sequencing, the total length of the target regions is about 50 Mb, whereas in the TargetEC method, which also covers CNSs, the total length of the target regions is about 150 Mb and they are scattered over the chromosomes. The sequence read data obtained by the TargetEC method applied to the PVG/Seac inbred strain (accession number: DRA004543) , which was crossed with a fancy rat to establish the KRFS4/Kyo inbred strain  and does not possess head spot phenotype, was used as a control. The sequence read data obtained by the TargetEC method applied to other 20 rat inbred strains (BDIX/NemOda, BDIX.Cg-Tal/NemOda, BN/SsNSlc, BUF/MNa, DOB/Oda, F344/DuCrlCrlj, F344/Jcl, F344/NSlc, F344/Stm, HTX/Kyo, HWY/Slc, IS/Kyo, IS-Tlk/Kyo, KFRS3B/Kyo, LE/Stm, LEC/Tj, NIG-III/Hok, RCS/Kyo, ZF, ZFDM) (accession number: DRA004543) , non of which possess head spot phenotype, was used to filter common variants.
Quality control of the sequencing reads and mapping
A detailed description of the analysis of the sequence reads is available elsewhere . In brief, the reads generated using the Illumina HiSeq platform were subjected to quality control to exclude low-quality reads. We used the FastQC program (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) to evaluate the overall read quality. We trimmed reads from the 3′-end to address low-quality bases (quality score of <30). After this trimming step, shortened reads (length of <30) were discarded from further analysis. Then, the reads were mapped to the rat genome assembly rn5 (RGSC 5.0, March 2012), which was downloaded from the UCSC Genome Browser , by using BWA (v0.7.4)  with the default parameters. SAMtools (v0.1.12a) , Picard tools (v1.87) (http://broadinstitute.github.io/picard/), and the Genome Analysis Toolkit (GATK; v3.6)  were used for post-processing of the mapped reads .
Identification and annotation of variants
Variant calling was conducted using the HaplotypeCaller utility in GATK. ANNOVAR (version 22-March-2015)  was used for the functional annotation of variants, with RefSeq genes and Ensembl genes as the rat gene annotations.
Genome data analysis
The Integrative Genomics Viewer (IGV; v2.2.13)  was used to visualize the mapped reads and variants. The UCSC Genome Browser was used for the comparative analysis of genomes and their associated annotations . Spatial proximity on the chromosome was analyzed using the ChromContact web server , which uses high-resolution Hi-C data for human cell lines .
Identification of a candidate mutation for the head spot phenotype in KFRS4/Kyo
First, we compared the 211 SNVs with those identified in the PVG/Seac inbred strain , which is the one used to establish the KFRS4/Kyo strain by crossing with a fancy rat , and in other 20 inbred rat strains (BDIX/NemOda, BDIX.Cg-Tal/NemOda, BN/SsNSlc, BUF/MNa, DOB/Oda, F344/DuCrlCrlj, F344/Jcl, F344/NSlc, F344/Stm, HTX/Kyo, HWY/Slc, IS/Kyo, IS-Tlk/Kyo, KFRS3B/Kyo, LE/Stm, LEC/Tj, NIG-III/Hok, RCS/Kyo, ZF, ZFDM) . None of these 21 strains possess head spot phenotype. From the 211 SNVs, we filtered out those SNVs that were commonly observed in at least one of the 21 inbred strains. This filtering drastically reduced the candidate SNVs, and only one SNV remained as a KFRS4/Kyo-specific SNV (Additional file 1: Table S1). This SNV (chr15: 93,740,390 A → G) does not seem to be a causative mutation for the phenotype, because it is located in an intergenic region with low conservation among vertebrates (phastCons score = 0.000). Although the TargetEC method is designed to capture exons and CNSs, such region can be a target because a shorter region must be expanded to at least 100 bp in capture probe design to ensure efficient capture of the region . Next, we compared the 58 indels with those identified in the 21 strains. After filtering out the commonly observed indels, 10 indels remained as KFRS4/Kyo-specific indels (Additional file 1: Table S1). All of these indels do not seem to be causative mutations for the phenotype, because they are located in intergenic regions with either low conservation or indels also in other species. As reported previously , and also confirmed in this study, there were no KFRS4/Kyo-specific SNVs or indels on Ednrb, one of the genes residing within the genomic interval identified by the linkage analysis and suspected to be a candidate gene for its involvement in the particular coat color phenotypes observed in other animals [8, 9] as well as in rats [5–7].
Epigenetic features in the deleted genomic region
For the deleted region, the corresponding region in humans was found at chr13:78.5–78.6 Mb (hg19 assembly) (Fig. 3). The sequence identity in this region between rat and human genomes is 38.5%. The relative position and orientation of Ednrb are also similar between rats and humans. Because the CNSs in rat, for which we designed the probes for the TargetEC method, are highly conserved among vertebrates, five of the six CNSs in the deleted sequence have their counterparts in the syntenic region in human. The sequence identity for each of the CNSs between rat and human genomes ranges from 95.2 to 99.8%. From the epigenetic features, we found two sequence regions that seem to be enhancers, characterized by prominent peaks of H3K4me1, H3K27ac, and DNase I hypersensitive sites, within the region that corresponds to the deleted region in KFRS4/Kyo. One of the putative enhancers is located in the corresponding CNSs in rat, indicating that it might also work as an enhancer in rat.
Possible three-dimensional (3D) interaction between the candidate enhancer and the promoter of Ednrb
To further confirm whether some of the putative enhancers identified in the deleted CNSs correspond to distant enhancers of Ednrb, we analyzed possible 3D interaction of the chromosome between the putative enhancers and the transcription start site (TSS) of Ednrb. We used Hi-C data for this analysis. Hi-C is a method for studying 3D chromatin interactions by cross-linking spatially proximal genomic fragments followed by high-throughput paired-end DNA sequencing . It has been successfully applied to identify long-range enhancer-promoter interactions . Although Hi-C data for rats are not yet available, we transferred the genomic coordinates for rats to those for humans and inferred the 3D interactions in rats because it has been shown that overall topology does not differ significantly between distinct cell types and even between species [30, 31]. By using the ChromContact web server , which visualizes Hi-C data in a 4C-like representation by specifying a position of interest as an anchor, we plotted a contact profile of spatial proximity to the TSS of Ednrb (Fig. 3). The resolution of Hi-C experiments has improved to a maximum of the kilobase order in recent years , and we used a 10-kb resolution in the present study. As shown in the contact profile, the putative enhancer at the CNS is located within a peak of the contact profile, indicating a possible interaction between TSS of Ednrb (i.e., anchor) and the putative enhancer, although the distance between them in terms of the genomic sequence is approximately 100 kb. This further supports the hypothesis that the putative enhancer in the deleted genomic region might be a melanocyte-specific enhancer of Ednrb.
We also analyzed the epigenomic features of the corresponding genomic region in mouse by using the Virtual 4C web application (http://promoter.bx.psu.edu/hi-c/virtual4c.php) (Additional file 4: Figure S3). We used the data for CH12 cell line, which is derived from B-cell lymphoma, because this is the only cell line with high resolution Hi-C data (5 kb resolution) in mouse. Although the Hi-C signal is only weakly support the interaction between the predicted enhancer and the promoter of Ednrb, DHS Linkage , which is the prediction of the interaction between a pair of genomic regions based on the correlation of DNase I hypersensitivity site data from various cell types, indicates that the predicted enhancer might interact with the promoter of Ednrb. A peak for H3K4me1 is also assigned to the predicted enhancer in CH12 cell line. These data in mouse further support that the deleted genomic region might act as an enhancer of Ednrb.
In the present study, we identified a deletion that is predicted to be causative of the head spot phenotype of the KFRS4/Kyo rat strain in the genomic interval determined by linkage analysis for this phenotype . This deletion is located upstream of Ednrb, one of the genes responsible for Waardenburg syndrome type 4 (WS4), also known as Waardenburg-Shah syndrome, characterized by hypopigmentation of hair, skin, and eyes, congenital sensorineural deafness, and Hirschsprung disease, which exhibits aganglionic megacolon . The involvement of Ednrb in WS4 is explained by its function in the development and migration of enteric neurons and melanocytes, both of which originate from neural crest cells. It has been revealed that SOX10 acts as an upstream transcription factor of Ednrb in the development of enteric neurons ; however, it seems that SOX10 is not involved in the regulation of Ednrb expression during melanocyte development [33, 34] and it is suggested that there might be other upstream factors of Ednrb expression for the development and migration of melanocytes [33, 35]. The KFRS4/Kyo strain possesses a head spot phenotype, but not aganglionic megacolon. This suggests that the head spot phenotype observed in KFRS4/Kyo is not due to a defect in the gene body of Ednrb, but is caused by a mutation in a regulatory region specific for the differentiation and migration of melanocytes. Indeed, the gene structure of Ednrb itself does not have any mutations. From these observations, it is strongly suggested that the deletion identified upstream of Ednrb might contain a melanocyte-specific enhancer that is responsible for the head spot phenotype of KFRS4/Kyo.
Although the lines of evidence presented in this study strongly suggest that the deletion found upstream of Ednrb contains a melanocyte-specific enhancer of the gene, direct experimental proof of this is still required to conclusively show its functional involvement. One possibility for obtaining such proof is qRT-PCR to quantify the difference in expression level between the wild type and the mutant strains. However, it will be difficult to detect any significant difference in the Ednrb expression level because the head spot phenotype is focal to a specific area. Moreover, EDNRB signaling is thought to be required only in a certain period in the early developmental stage, in which neural crest cells differentiate into melanocytes that migrate to the epidermis . Another possible strategy for obtaining a direct experimental proof is to conduct a reporter assay in which the predicted melanocyte-specific enhancer is cloned upstream of a reporter gene or to employ a genome editing technique to disrupt the predicted melanocyte-specific enhancer in the wild type to see if a similar phenotype, namely, a head spot, is observed. Yet another possibility, albeit one that would not provide a direct proof, is to search for other strains or species with a similar phenotype having a mutation in the corresponding genomic region of the predicted enhancer. This would provide strong supporting evidence for the involvement of the predicted enhancer in the development of melanocytes.
Mutations in coding regions may have severe phenotypic effects such as embryonic lethality because, for example, a non-synonymous substitution can lead to the production of proteins without activity or a nonsense mutation can be degraded by nonsense-mediated mRNA decay or produces a truncated protein product. On the other hand, mutations in non-coding regulatory regions, although these have not been studied well compared with those in coding regions, might have milder effects because such mutations sometimes change only the spatio-temporal expression patterns in a certain tissue or in a developmental stage. As we show in this study, the head spot phenotype in the KFRS4/Kyo rat strain is one such phenotype because Ednrb-null mutants usually possess both white spots and megacolon, which is a characteristic phenotype of WS4 due to the absence of enteric neurons. Phenotypic effects of the mutations in CNSs are not well documented so far compared with those in protein coding regions, while the fact that the total length of CNSs in a mammalian genome is almost comparable to that of exons  leads us to speculate that there must be a number of regulatory mutations that affect various phenotypes. The TargetEC method , from which the data used in this study were obtained, can be efficiently applied to identify such regulatory mutations.
Although it is rather straightforward to infer the functional consequence of mutations in gene bodies, it is often difficult to interpret the effect of regulatory mutations, especially if they are located far from their putative target genes. As shown in this study, the efficient use of information about the epigenetic status of genomes, as well as comparative genomic analysis, helps to confirm the functional implications of such regulatory mutations. Epigenomic information, which is rapidly expanding with the progress of several international projects [14, 15], together with information about the three-dimensional conformation of chromatin , will shed more light on the detailed regulatory networks of genes.
We successfully identified a candidate mutation by using the data obtained from our recently devised method, TargetEC, which can detect mutations not only in exons but also in CNSs , together with comparative genomic analysis of publicly available epigenomic data. Based on the success rate of exome sequencing studies  and the proportion of CNSs, which are thought to be conserved because of their functional importance, there might be a significant number of mutations causative of certain genetic disorders in regulatory regions. By systematically identifying these mutations in genetic disorders, we will be able to deepen our understanding of the regulatory mechanisms of gene expression and disease etiology, which should lead to the development of mechanism-based therapies.
Conserved non-coding sequence
Single nucleotide variant
Waardenburg syndrome type 4
We thank Mayuko Yokoe for her assistance in the PCR experiments. We are also thankful to the National BioResource Project - Rat (http://www.anim.med.kyoto-u.ac.jp/nbr/) for providing rat strains used in this study.
This work was supported in part by the Cooperative Research Project Program of the Medical Institute of Bioregulation, Kyushu University, to OO, and the Genome Information Upgrading Program of the National BioResource Project, Japan Agency for Medical Research and Development, to OO, TK, and MS.
Availability of data and materials
The TargetEC probe set is commercially available from Roche NimbleGen under the design name 140929_RN5_MS_EZ_HX1. The sequence data are available at DDBJ Sequence Read Archive (accession number: DRA004543).
MY and MS conceived and designed the study. TK and OO conducted the experiments. MY, TS, DS, and MS performed the analyses. MY and MS wrote the manuscript with the help from all of the authors. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
All animal experimentation protocols were approved by the Institutional Animal Care and Use Committees of Kyoto University (approval No. MedKyo15001) and were conducted according to the Regulation on Animal Experimentation at Kyoto University.
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