BACKGROUND
the high prevalence of obesity and type  <dig> diabetes is a serious public health issue that is associated with devastating health consequences such as cardiovascular diseases  <cit> .
the world health organization estimated that more than 10 % of the world’s adult population  was obese  in  <dig>  and that, in  <dig>   <dig> million people had diabetes, 90 % of whom had type  <dig> diabetes .
genetic predisposition is recognized as a major risk factor for the development of obesity and type  <dig> diabetes; estimates of the heritability range from 50 % to 60 % for both diseases  <cit> .
therefore, identification of underlying susceptibility genes would define potential targets and pathways for intervention and treatment of obesity and type  <dig> diabetes.
the genetics of human obesity and type  <dig> diabetes is complex  <cit> .
it involves multiple susceptibility genes and their interactions with environmental factors  <cit> .
animal models that share both physiologic and genetic similarity with humans are used in obesity and type  <dig> diabetes research to minimize the confounding effects of heritability, genetic heterogeneity and environment that determine these diseases in humans  <cit> .
the tallyho/jng  mouse is a polygenic inbred model for human obesity and type  <dig> diabetes  <cit> .
th mice manifest many of the disease phenotypes observed in human obesity and type  <dig> diabetes including hyperleptinemia, hyperinsulinemia, insulin resistance, glucose intolerance, hyperlipidemia, and hyperglycemia  <cit> .
these mice also exhibit increased islet insulin secretion in response to glucose and β-cell mass  <cit> .
the genetic basis for obesity and type  <dig> diabetes in th mice has been studied using outcross experiments with normal strains which has led to the identification of multiple quantitative trait loci  linked to adiposity and hyperglycemia  <cit> .
in addition to genetic analysis, the th mouse has been used in the development of therapeutic agents for obesity and type  <dig> diabetes  <cit>  and has also served as a model system for many diabetes and obesity related abnormalities, including decreased exercise capacity  <cit> , impaired wound healing , periodontitis  <cit> , tissue susceptibility to hypoxia  <cit> , bone loss  <cit> , circadian disruption  <cit> , and vasculature abnormalities .
in this study, we sequenced the whole genome of the th mouse using next-generation sequencing with the goal of identifying genome-wide sets of single nucleotide polymorphisms  and small insertions and deletions , a subset of which are present exclusively in the th mouse .
a complete catalogue of th genomic variants  will aid the identification of causative variants and genes underlying the diseases observed in th mice, which will maximize the relevancy of this model for human obesity and type  <dig> diabetes.
knowing the distribution of variants across the genome of the th mouse vs. other classic and wild-derived inbred strains  <cit>  may identify naturally occurring gene variant sets that can represent model genes for the complex human obesity and type  <dig> diabetes.
as a proof of principle, we integrated our genome variant data with genetic mapping data and identified a variant in cell death-inducing dffa-like effector c  whose effect on protein function we characterized.
methods
description and origin of the th mouse strain
the th mouse is originally derived from an outbred colony of the theiler original strain in which two male mice spontaneously became polyuric, glucosuric, hyperinsulinemic, and hyperglycemic   <cit> .
in  <dig>  male diabetic progeny and apparently normal female mice were imported into the jackson laboratory   and an inbred strain was then established by selecting for male hyperglycemia by dr. jürgen naggert’s research group at tjl.
in  <dig>  a sub-colony was initiated in our laboratory with breeding pairs from dr. naggert’s research colony  <cit>  and used in this study.
after arrival at our laboratory, we interbred siblings for  <dig> generations followed by one generation backcrossing and an additional  <dig> generations of sibling interbreeding.
since the generation prior to arrival at our laboratory remains unknown, we refer to the generation used for whole genome sequencing in this study as f?
+ f18n1f <dig>  all animal studies were carried out with the approval of marshall university animal care and use committee.
dna sequencing, read alignment, and bioinformatics
high quality genomic dna was extracted from the liver of a male th mouse from our colony using a qiagen  genomic-tip 100/g kit.
genomic dna  was sheared using a covaris  s <dig> instrument to ~350 bp and used to construct a sequencing library using an illumina  dna sample preparation kit according to the manufacturer’s recommended protocol.
the library was quantified using thermofisher  qubit fluorimetry and sized on an agilent  bioanalyzer dna chip.
the resulting whole genome library  was amplified on a flow cell using an illumina cbot cluster station and then sequenced on an illumina hiseq  <dig> in the marshall university genomics core facility.
three sequencing runs  were performed in order to obtain adequate depth of coverage.
sequencing reads were aligned to the c57bl/6j  reference genome  using bowtie <dig> v <dig> .
<dig>  <cit> .
our variant calling pipeline was based on that developed by wong et al.
<cit>  to call variants in the fvb/nj mouse strain.
briefly, duplicate reads were removed with samtools v <dig> .
<dig>  <cit>  using the “samtools rmdup” command.
to improve quality of the variant calling, local realignment around insertions and deletions was performed using gatk v <dig> .
<dig>  <cit>  by first running the “realignertargetcreator” command and then the “indelrealigner” command.
snps and indels were called by generating a pileup using the “samtools pileup” command with options ueds, and piping the results to the “bcftools view” command with options “-p  <dig>  –vbcgn”.
variants were filtered using the vcftools package  <cit>  version  <dig> .
<dig>  in order to maintain maximal consistency with the mouse genome project   <cit> , we used the same options as in wong et al.
<cit> .
we identified variants in the th strain that did not occur in any of the  <dig> mouse strains published in the mgp , which we term “private” variants, following keane et al.
<cit> .
to do this, we generated a list of all genomic locations of th variants in the form of a bed file, and then performed variant calling on the bam files for the  <dig> mgp mouse strains at those locations, using samtools’ “–l” option along with the same parameters as were used to call th variants.
th snps qualified as private if no mgp strain had the same snp at the same location, where at least  <dig> of the  <dig> strains had a call quality at least  <dig> and read depth at least  <dig> at that location.
th indels qualified as private if no mgp strain had a variant at the same genomic location, with the same criteria for call quality and read depth being applied.
we then added functional consequence annotation, including “sorting intolerant from tolerant”   <cit>  prediction of protein function changes of coding variants, using a local installation of ensembl variant effect predictor  version  <dig>  <cit> .
output from vep includes classification of each variant using a set of one or more sequence ontology  terms  <cit> .
variants are classified multiple times if they lie within a region intersecting multiple known transcripts.
we tabulated the number of variants according to their collection of so terms, collecting into a single “multiple classification sets” group all variants that had multiple, distinct sets of so terms due to their presence in multiple transcripts.
since this latter group is large, and may include potentially interesting variants, we identified a set of ten so terms we call “potentially pathogenic”: frameshift_variant, inframe_deletion, inframe_insertion, missense_variant, stop_gained, stop_lost, initiator_codon_variant, splice_region_variant, splice_acceptor_variant, and splice_donor_variant.
we tabulated the number of variants in the “multiple classification sets” that classified with each of these so terms for one or more transcripts.
the same classification was performed on the private variants.
for missense variants, we additionally used protein variation effect analyzer v  <dig>    <cit>  to provide further prediction of the functional effects of protein changes.
in order to uniquely associate a representative so term with each variant, we chose a single representative so term for each distinct set of so terms identified for a given variant in a given transcript, as shown in additional file 2: table s <dig> and additional file 3: table s <dig>  for example,  <dig> snps were classified with the two so terms “intron_variant” and “splice_region_variant”: for these snps we chose “splice_region_variant” as the representative term.
using this process, variants occurring in multiple transcripts that had multiple, distinct sets of so terms associated with them, resulted in having multiple representative so terms.
for such variants, we chose the “most pathogenic” representative so term, ordering them in the following priority: “stop_gained”, “stop_lost”, “frameshift_variant”, “missense_variant”, “inframe_insertion”, “inframe_deletion”, “splice_acceptor_variant”, “splice_donor_variant”, “splice_region_variant”, “initiator_codon_variant”, “synonymous_variant”, “5_prime_utr_variant”, “3_prime_utr_variant”, “mature_mirna_variant”, “upstream_gene_variant”, “downstream_gene_variant”, “intron_variant”, “non_coding_transcript_variant”, “intergenic_variant”.
in order to compare our variant sets to known associations between genetic variants and human traits for which th is a model, we first retrieved the genome wide association study  catalog  <cit>  from the european bioinformatics institute  , and filtered the catalog by the column “disease/trait” for each of the terms “obesity”, “diabetes”, and “metabolic”.
for each filtered catalog, we extracted all human entrez geneids listed in the columns mapped gene, upstream_gene_id, and downstream_gene_id.
using the ensembl interface to biomart  <cit> , we converted these entrez geneids to ensembl human gene ids.
additionally, we retrieved a list of genes from pigeyre et al.
<cit>  in which mutations have been previously shown to cause a monogenic obesity phenotype in humans, and found ensembl ids for these genes using the same tool.
for all these human ensembl gene ids, we found ensembl mouse gene ids of orthologous genes, again using the ensembl interface to biomart.
we then filtered our variant sets  to identify potentially pathogenic variants associated with each of these orthologous gene sets.
aligned reads were uploaded to the sequence read archive  at the national center for biotechnology information  and can be accessed via accession number srp <dig>
cell culture, plasmids, transfections, and microscopy
cos- <dig> cells  were grown in dulbecco’s modified eagle’s medium with l-glutamine,  <dig>  g/l glucose and sodium pyruvate  supplemented with 10 %  bovine serum  and  <dig> units/ml penicillin and 100 mg/ml streptomycin .
the murine cidec cdna was procured from genscript .
a green fluorescent protein fusion, pacgfp1-cidec was created by cloning the cidec cdna with 5′ hindiii and 3′ bamhi sites upstream and in-frame with the acgfp <dig> sequence in pacgfp1-n <dig> .
the entire cidec coding region was included except for the stop codon  <cit> .
site-directed mutagenesis was used to introduce the missense polymorphism r46s .
plasmids, pacgfp1-cidec  and pacgfp1-cidec , were transfected into cos- <dig> cells cultured on 4-well chamber slides   using lipofectamine ltx&plus  following the manufacturer’s instruction  <cit> .
cos cells do not express endogenous cidec  <cit> .
after transfection, the cells were cultured for 48 h with and without 400 μm bsa-complexed oleic acid   <cit> .
cells were then washed with pbs, fixed by 4 % paraformaldehyde for 30 min, and permeabilized in  <dig>  %  saponin  in pbs for 20 min.
lipids were stained with 1 μg/ml nile red  which partitions with neutral lipids; cells were washed with pbs .
nuclei were labeled by placing coverslips onto slides with prolong® gold antifade mountant with dapi .
coverslips were dried and cells viewed on a leica sp <dig> confocal microscope located in the marshall university imaging center.
each experiment was performed in quadruplicate samples.
association of full length cidec with lipid droplets in cos- <dig> cells was assessed using imagej software by performing a co-localization analysis of gfp florescence with nile red labeled lipid droplets in each z-section of a transfected cell  <cit> ; these results were then averaged to calculate the overall co-localization in a given cell .
the data were from  <dig> cell clusters in each condition from two independent experiments.
quantitative data were presented as means ± sem.
two-tailed student’s t-tests were performed on data.
differences of p <  <dig>  were considered significant.
RESULTS
identification of snps and indels
the whole genome of the th mouse was sequenced to an average depth of ~ <dig> x coverage using data from three illumina paired-end read sequencing runs.
the sequencing reads were mapped to the b <dig> mouse reference genome .
using a samtools-based pipeline  <cit> , we identified  <dig> , <dig> snps  and  <dig> , <dig> indels  genome-wide, relative to the reference strain c57bl/ <dig> .
the positional distribution of variants on each chromosome is shown in fig.
<dig>  to assess the reliability of our data, pcr amplification and sanger sequencing were applied to  <dig> homozygous snps to determine whether they agreed with next generation sequencing results .
we found that all  <dig> snps were consistent with the illumina sequencing data.fig.
<dig> distribution of detected variants on the th genome.
total number of variants , number of snps , and number of indels  per megabase as a function of genomic position.
these were calculated by sliding a one-megabase window across each chromosome in steps of one kilobase and totaling the number of variants in the window
we observed a small percentage of heterozygous snps and indels in the th genome .
possible explanations for the apparent presence of heterozygosity include errors in variant calling, true residual heterozygosity, and recent heterozygous mutations not yet fixed to homozygosity  <cit> .
we did not consider heterozygous snps and indels in our subsequent analyses.
functional consequences of snps and indels
we assigned putative functional consequences to the set of homozygous variants  in th using variant effect predictor   <cit> , which classified the variants using terms from the sequence ontology   <cit>  .
variants which intersected multiple transcripts, and for which the set of so terms associated with the variant differs among those transcripts, were categorized as “multiple classification sets”.
for each of ten so terms we designated “potentially pathogenic” , we counted the number of variants in the “multiple classification sets” category that classified under that so term in one or more transcripts.
for each distinct collection of so terms identified for a variant, we chose a representative term .
for variants with multiple classification sets this potentially resulted in multiple representative so terms: for these variants with multiple classifications we categorized the variant as the “most pathogenic” according to the prioritization defined in the methods.
this resulted in a unique so term for each variant: the distributions of these so terms among snps and indels are shown in figs.
<dig> and  <dig>  respectively.
the majority of snps were either intergenic  or intronic .
the next two largest snp groups were those which were located 5 kb upstream  or downstream  from a coding gene.
a small number of snps  were located within the protein coding regions of gene bodies.
snp variants resulted in  <dig>  synonymous codon changes,  <dig>  non-synonymous codon changes,  <dig> conversions of coding codons to stop codons and  <dig> conversions of stop codons to coding codons.fig.
<dig> classification of snps by sequence ontology term.
the large pie chart shows the characterization of all tallyho/jng snps relative to the c57bl/ <dig> reference genome; the smaller chart shows the distribution of the subset of these we term “potentially pathogenic”, as described in the methods
fig.
<dig> classification of indels by sequence ontology term.
the large pie chart shows the characterization of all tallyho /jng indels relative to the c57bl/ <dig> reference genome; the smaller chart shows the distribution of the subset of these we term “potentially pathogenic”, as described in the methods
there were  <dig>  snps causing non-synonymous changes to one or more protein-coding transcripts in  <dig>  genes.
we evaluated the effect of the amino acid substitutions resulting from the snps using sift  <cit>   and provean  <cit> .
these algorithms classify the changes as either deleterious  or tolerated/neutral .
sift classified  <dig>  % of the non-synonymous substitutions  in  <dig>  genes as deleterious.
provean classified  <dig>  %  in  <dig> genes as deleterious.
<dig>  % of the missense variants  in  <dig> genes were classified as deleterious by both algorithms.
as expected, the vast majority  of th indels fell into noncoding groups .
the total in-frame and frameshift indels were  <dig> and  <dig>  respectively .
proportions of snps and indels in each functional class are similar to those found in the fvb/nj genome  <cit>  .table  <dig> comparison of distribution of variants in tallyho/jng to that in fvb/nj
comparison to the human genome-wide association study catalog and to known mendelian obesity genes
in order to interpret our data in the context of human disease, we collected human gene ids in which variants were associated with obesity, diabetes, or metabolic syndrome, from the ebi gwas catalog version  <dig> .
<dig>  <cit> .
these gene ids were mapped to orthologous mouse ensembl gene ids using the ensembl interface to biomart  <cit> , generating sets of orthologous mouse genes.
we additionally collected a list of known mendelian obesity genes in humans from pigeyre et al.
<cit> , and generated a set of mouse orthologous genes as aforementioned.
we then filtered the potentially pathogenic th variants against these gene sets to provide lists of potentially pathogenic variants for each of these diseases.
we found  <dig> genes with potentially pathogenic variants in the gwas obesity gene set,  <dig> in the gwas diabetes gene set, and  <dig> in the gwas metabolic gene set.
additionally, we found  <dig> genes with potentially pathogenic variants orthologous to genes known to cause monogenic syndromic obesity, four orthologous to genes known to cause monogenic non-syndromic obesity, and three orthologous to genes known to cause monogenic non-syndromic lipodystrophy .table  <dig> variants in genes linked to traits of interest by gwas or in mendelian obesity genes
th private snps and indels
we identified variants that were present in the th strain and absent from  <dig> mgp strains by passing the bam files for each strain through our variant-calling pipeline at each location where we identified a th variant.
a th snp was identified as “private” if it was not called as the same snp in any of the other  <dig> strains, and a th indel was identified as “private” is no variant was identified at the same location in any of the other  <dig> strains.
additionally, the designation of “private” required a call quality at least  <dig> and read depth at least  <dig> in at least  <dig> of the  <dig> strains.
in this analysis, we identified a total of  <dig>  private snps and  <dig>  private indels, which represented  <dig>  % and  <dig>  % of the total snps and indels, respectively.
we classified private variants by so term using vep as described in the analysis for all variants above.
of the  <dig>  private snps,  <dig> were missense substitutions, four resulted in the gain of a stop codon, and one in the loss of a stop codon .
among the  <dig>  private indels,  <dig> were frameshift indels and  <dig> were in-frame indels .
for these private snps and indels, along with those associated by vep with splice regions and initiator codons, we counted the number of such variants occurring in each gene.
there were  <dig> private snps meeting one or more of these criteria in  <dig> genes  and  <dig> private indels meeting one or more of these criteria in  <dig> genes .
for the private snps, we found that  <dig> of the  <dig> genes had one or more private snps determined by sift to be deleterious missense variants,  <dig> had one or more private snps determined by provean to be deleterious, with  <dig> genes having snps determined to be deleterious by both algorithms.fig.
<dig> classification of private snps by sequence ontology term.
the large pie chart shows the characterization of all tallyho/jng snps relative to the c57bl/ <dig> reference genome that are not present in  <dig> previously sequenced strains; the smaller chart shows the distribution of the subset of these we term “potentially pathogenic”, as described in the methods
fig.
<dig> classification of private indels by sequence ontology term.
the large pie chart shows the characterization of all tallyho/jng indels relative to the c57bl/ <dig> reference genome that are not present in  <dig> previously sequenced strains; the smaller chart shows the distribution of the subset of these we term “potentially pathogenic”, as described in the methods
in the analysis linking our variant lists with human gwas studies and human mendelian obesity genes described above, we determined the number of variants in orthologous genes which were private to the th mouse .
characterization of the tabw <dig> obesity qtl interval on chromosome 6
tabw <dig>  is a major obesity qtl identified in th mice, and confirmed by a congenic strain on the b <dig> background  <cit> .
using subcongenic analysis we have determined that the effect of tabw <dig> on obesity could be attributed to two adjacent loci, tabw2a and tabw2b  <cit> .
there are  <dig> and  <dig> protein coding genes cataloged in the ensembl mouse annotation database in the tabw2a  and tabw2b  intervals, respectively.
a total of  <dig>  snps and  <dig>  indels were found in the tabw2a and tabw2b intervals, of which  <dig>  snps and  <dig>  indels were in protein-coding genes .
among those, only  <dig> snps  in the tabw2a interval and  <dig> snps  in the tabw2b interval were classified as deleterious by both sift and provean .
we then conducted a literature review of the biological function of the  <dig> genes in the tabw <dig> interval with snps classified as deleterious by either algorithm, searching for potential link to obesity .
from this search, the cidec gene, containing a snp identified as deleterious by both sift and provean, drew our attention.
cidec is a lipid droplet protein that is involved in the regulation of cellular lipid droplet size and lipid storage during lipid metabolism in adipocytes  <cit> .
a loss of function mutation in cidec causes a familial partial lipodystrophy  <cit> .
further, weight loss via a low calorie diet was correlated with a reduced gene expression of cidec in adipose tissue in humans  <cit> .table  <dig> numbers of variants in the tabw <dig> locus
tabw2a
tabw2b
gcfc2
81941369
cgg/tgg
r/w
7/17
0
- <dig>
ccdc142
htra2
mogs
rtkn
1700003e16rik
tet3
aak1
gfpt1
d6ertd527e
chchd6
89467434
cgt/tgt
r/c
5/8
<dig>
- <dig>
zfyve20
lmod3
ttll3
cidec
113433131
cgt/agt
r/s
3/6
0
- <dig>
irak2
rpl28-ps4
ret
118179286
acc/atc
t/i
5/20
0
- <dig>
cacna1c
cecr5
a2m
121661523
acg/aag
t/k
21/36
0
- <dig>
8430419l09rik
atf7ip
bc049715
136840191
atg/acg
m/t
1/1
<dig>
- <dig>
art4
mgp
ptpro
igbp1b
pik3c2g
139967890
agt/tgt
s/c
16/16
<dig>
- <dig>
chr chromosome, a.a. amino acid, ref reference, th tallyho
snps predicted to be deleterious by both sift and provean are shown in bold
there was one nucleotide substitution in the cidec coding sequence,  <dig> c > a. the substitution of  <dig> c > a resulted in an amino acid difference of r46s  between b <dig> and th strains .
to examine the function of the cidec r46s polymorphism, we created murine plasmids acgfp1-cidec  and acgfp1-cidec  and transiently transfected them into cos- <dig> cells.
cos- <dig> cells have been used as models for monitoring cidec function in prior studies because they lack endogenous cidec activity .
it was apparent that acgfp1-cidec was localized to distinct lipid droplets stained with nile red .
when we considered nile red lipid staining specifically associated with gfp fluorescence , cos- <dig> cells transfected with the s <dig> variant exhibited an increase in lipid accumulation compared to cells transfected with the wild type r <dig> variant in the media both with and without oleic acid .
we speculate that the s <dig> variant may be hypermorphic in that it enhances the function of cidec in promoting lipid accumulation in lipid droplets.fig.
<dig> representative images of lipid droplets in cos- <dig> cells transfected with  acgfp1-cidec  and  acgfp1-cidec .
a and c present cells cultured in media without oleic acid  and b and d present cells cultured in media with oa.
images of nuclei labeled with dapi, neutral lipid labeled with nile red and location of acgfp <dig> were merged.
e quantification of lipid specifically associated with gfp in cells.
using imagej software, the ratio of nile red/gfp signal was calculated from  <dig> lipid droplet clusters each for r <dig> and s <dig> without oa and  <dig> lipid droplet clusters for r <dig> and  <dig> lipid droplet clusters for s <dig> with oa.
data are presented as means ± sem.
*p <  <dig>
discussion
landscape of the tallyho genome
we found a total of  <dig> , <dig> variants  in the th genome, compared to the reference b <dig> genome .
as expected, the large majority of variants occurred in non-protein coding regions of the genome.
the number of snps was broadly consistent with that reported by keane et al.
and wong et al.
for  <dig> laboratory strains  <cit> , and substantially fewer than the four wild-derived strains  sequenced in the keane et al.
study  <cit> .
we found  <dig>  % to  <dig>  % more indels when compared to those  <dig> laboratory strains, and again considerably fewer than the four wild-derived strains.
the proportion of private snps we observed was higher than  <dig> of the laboratory strains from keane et al.
<cit> , with the two exceptions being the two strains nod/shiltj and nzo/hiltj.
we also showed a lower proportion of private snps in the th strain than discovered in fvb/nj  <cit> .
the proportion of private indels we observed in th was higher than in these studies.
although the origins of the theiler original mouse are unknown, the generally higher proportion of private variants in th  is consistent with the possibility that th has a unique ancestor.
we also note that our sequencing coverage of  <dig> x was higher than in these other studies, increasing our general power to detect variants.
classification of variants
we provided a comprehensive catalog of variants of the th mouse, relative to the b <dig> mouse, classified according to the effect of the variant on protein sequence.
these effects were predicted by comparing the location of the variant on the reference genome to known genomic annotations provided by the ensembl gene database  <cit> .
while it is relatively straightforward to automate the classification of a large number of variants via genomic annotations on a coarse scale, providing detailed interpretations of the effect of some mutations requires greater inspection of the context of the mutation that cannot be readily automated and performed on a genome-wide scale.
for example, the effect of intronic mutations is not well characterized or understood at present.
our aims in this study include both providing a general overview of the landscape of the th mouse genome relative to the reference genome, and also providing sufficient detail of the nature of the  <dig>  million variants discovered.
to this end, we used an automated process to classify variants into a large number of precisely defined categories, and then subsequently reduced the number of these categories, subjectively categorizing them into a smaller set of representative terms.
the first categorization provides a fine-grained resource for searching for individual variants that may have phenotypic consequences, and the second provides a general overview of the landscape of the th genome.
the sequence ontology   <cit>  provides a controlled vocabulary for describing features and annotations associated with a biological sequence, including mutations at the sequence level, along with a formalized description of the relationships between these terms.
the variant effect predictor   <cit>  is a tool that predicts the effect of genomic variants by analyzing the variant in the context of annotations on the reference genomic sequence.
for each variant and each known transcript containing that variant, vep outputs a subset of so terms associated with that variant.
in many cases, this results in multiple so terms being associated with a single variant in the context of a single transcript.
for example, we found an a to g single nucleotide polymorphism  at genomic location  <dig> , <dig> on chromosome  <dig>  this snp occurs in the second nucleotide of the first codon of the second exon of the apolipoprotein b receptor gene, apobr, mutating the gac codon to ggc.
since this mutation results in a change in amino acid from aspartic acid to glycine, the snp is annotated with the so term “missense_variant”.
since this variant also occurs within three bases of a splice site, it is also annotated with the so term “splice_region_variant”.
in order to provide a complete, unbiased characterization of the types of variants discovered, we list all unique combinations of so terms for a single variant and the number of times those combinations occur .
in order to provide a concise overview of the relative proportions of occurrences of the major types of variants, for each unique combination of so terms we selected a “representative” so term .
these overviews are presented in figs.
<dig> and  <dig>
due to the presence of multiple transcripts for some genes, and due to the presence of overlapping genes, some individual variants are present in multiple transcripts.
in this case, vep outputs a potentially different combination of so terms for each of the transcripts in which the variant occurs.
when there are multiple, distinct, combinations for a given variant due to its presence in multiple transcripts, these variants are categorized in additional file 2: table s <dig> and additional file 3: table s <dig> as “multiple classification sets”.
in order to uniquely classify such variants in the overview , we picked a representative so term for each set of so terms using the strategy shown in additional file 2: table s <dig> and additional file 3: table s <dig>  and then chose the most pathogenic out of these representative so terms using the priority described in the methods.
filtering variants by the human gwas catalog
we employed a translational methodology to identify and prioritize candidate genes for complex disorders for which th is a model.
we examined all the th variants for potential link to relevant human disease using the ebi gwas catalog  <cit>  and generated enriched candidate gene lists for obesity, diabetes and metabolic syndrome.
we reviewed biological function for selected genes and identify genes we could connect to obesity and/or diabetes.
glis <dig> is a transcription factor that plays an important role in pancreatic development and insulin gene expression in beta cells  <cit> .
sorbs <dig> is involved in insulin stimulated glucose transport in adipocytes  <cit> .
igf2bp <dig> is a rna binding protein, participating in posttranscriptional rna processing, i.e., rna splicing, stabilization, transport, and translation  <cit> .
inactivation of the igf2bp <dig> gene caused a resistance to diet-induced obesity in mice  <cit> .
semaphorin 5a  is an axon regulator molecule and plays a role during neuronal and vascular development  <cit> .
collectively, our convergent approaches systematically integrate whole genome sequencing data and genetic information from gwas-derived findings and provide an opportunity to discover candidate genes for further functional validation.
missense polymorphism of r46s in cidec between b <dig> and th mice
among the genes with snps that were identified as deleterious by both sift and provean , were orthologous to mendelian lipodystrophy genes, and were contained within the tabw <dig> interval, we identified cidec, which is an important regulator of energy homeostasis directly involved in promoting the accumulation of triglyceride into intracellular lipid droplets  <cit> .
lipid droplets are spherical organelles found in many types of eukaryotic cells, including adipocytes, and are composed of a core of neutral lipids, such as sterol esters or triglycerides, surrounded by a monolayer of phospholipids, free cholesterol, and multiple specific proteins including cidec  <cit> .
in adipocytes, cellular energy is stored as triglycerides in lipid droplets, and in conditions of fatty acid excess, lipid droplets rapidly increase their volumes  <cit> .
the capacity of cidec in accumulating triglycerides into intracellular lipid droplets was demonstrated by transfection experiments using multiple cell types including c pre-adipocytes, 293t cells, and cos cells .
for example, transfection of full-length murine cidec into cos- <dig> cells increased total cellular triglycerides by 50 %  <cit> .
we found that the th allele of cidec s <dig> variant allowed for more lipid accumulation than the b <dig> allele of cidec r <dig> variant in cos- <dig> cells.
further functional validation of this missense polymorphism in vivo may provide an opportunity to understand the role of cidec in the context of obesity.
CONCLUSIONS
we have sequenced the whole genome of obese type  <dig> diabetic th mice by next-generation sequencing and generated a complete catalog of variants classified by location relative to genes and predicted consequences for protein products.
we filtered the list of variants to the tabw <dig> obesity qtl and identified a missense polymorphism in cidec whose protein function we characterized.
furthermore, by comparing the variant catalog to the human gwas catalog and to known human mendelian obesity genes, we were able to identify a list of susceptibility candidate genes that could be used to dissect the components of polygenic diseases such as obesity and type  <dig> diabetes.
additional files
additional file 1: table s <dig>  mouse genome project strains.
list of strains from the mouse genome project used in the determination of “private” variants.
additional file 2: table s <dig>  classification of th snps by sets of so terms associated with the snp.
additional file 3: table s <dig>  classification of th indels by sets of so terms associated with the indel.
additional file 4: table s <dig>  pcr primer sequences used for validation of selected variants.
additional file 5: table s <dig>  variants in orthologs of gwas-associated and mendelian genes.
list of orthologous genes to human genes associated by gwas studies to obesity, diabetes, and metabolic syndrome, and to mendelian obesity genes, with variant type and number.
additional file 6: table s <dig>  private snp classification.
classification of private th snps by sets of so terms associated with the snp.
additional file 7: table s <dig>  private indel classification.
classification of private th indels by sets of so terms associated with the indel.
additional file 8: table s <dig>  genes with pathogenic private snps.
list of genes with pathogenic private snps, with sift scores and so terms for the corresponding snp.
additional file 9: table s <dig>  genes with pathogenic private indels.
list of genes with pathogenic private indels, with so terms for the corresponding indel.
abbreviations
dnadeoxyribonucleic acid
ebieuropean bioinformatics institute
gwasgenome wide association study
indelinsertion and/or deletion
mbasemegabase
mgpmouse genome project
ncbinational center for biotechnology information
proveanprotein variation effect analyzer
qtlquantitative trait locus
siftsorting intolerant from tolerant
snpsingle nucleotide polymorphism
sosequence ontology
srasequence read archive
tabw2tallyho associated body weight 2
thtallyho/jng mouse strain
tjlthe jackson laboratory
vepvariant effect predictor
goran boskovic deceased
this work was supported in part by the nih/niddk-r01dk <dig>  nih/ncrr p <dig> rr <dig> and nih/nigms p20gm <dig> which funds the idea wv-inbre program and supports the marshall university genomics core facility and in part by aha-0855300e.
points of view in this document are those of the authors and do not necessarily represent the official position or views of the nih or the aha.
we thank kristy d. dillon and taryn p. stewart for maintaining mouse colonies, and david neff in the marshall university imaging core facility for his assistance in confocal microscopy.
availability of data and materials
raw sequencing data from this study are deposited in the national center for biotechnology information  sequencing read archive and are available via accession number srp <dig>
authors’ contributions
jd performed all data processing and bioinformatics, including alignment, variant calling, identification of private variants, and cross-referencing to the gwas catalog and mendelian obesity genes.
gb and jf prepared next-generation sequencing libraries and performed the sequencing.
jhk and jkp prepared genomic dna and conducted cell culture and transfection experiments and pcr.
jhk, jd and dap designed the overall research project and drafted the manuscript, tables and figures.
all authors except gb, who died unexpectedly during the course of the study, read and approved the final manuscript.
competing interest
the authors declare that they have no competing interests.

 the high prevalence of obesity and type diabetes is a serious public health issue that is associated with devastating health consequences such as cardiovascular diseases. the world health organization estimated that more than 10 % of the world’s adult population was obese in and that, in million people had diabetes, 90 % of whom had type diabetes. genetic predisposition is recognized as a major risk factor for the development of obesity and type diabetes; estimates of the heritability range from 50 % to 60 % for both diseases. therefore, identification of underlying susceptibility genes would define potential targets and pathways for intervention and treatment of obesity and type diabetes. the genetics of human obesity and type diabetes is complex. it involves multiple susceptibility genes and their interactions with environmental factors. animal models that share both physiologic and genetic similarity with humans are used in obesity and type diabetes research to minimize the confounding effects of heritability, genetic heterogeneity and environment that determine these diseases in humans. the tallyho/jng mouse is a polygenic inbred model for human obesity and type diabetes. th mice manifest many of the disease phenotypes observed in human obesity and type diabetes including hyperleptinemia, hyperinsulinemia, insulin resistance, glucose intolerance, hyperlipidemia, and hyperglycemia. these mice also exhibit increased islet insulin secretion in response to glucose and β-cell mass. the genetic basis for obesity and type diabetes in th mice has been studied using outcross experiments with normal strains which has led to the identification of multiple quantitative trait loci linked to adiposity and hyperglycemia. in addition to genetic analysis, the th mouse has been used in the development of therapeutic agents for obesity and type diabetes and has also served as a model system for many diabetes and obesity related abnormalities, including decreased exercise capacity, impaired wound healing, periodontitis, tissue susceptibility to hypoxia, bone loss, circadian disruption, and vasculature abnormalities. in this study, we sequenced the whole genome of the th mouse using next-generation sequencing with the goal of identifying genome-wide sets of single nucleotide polymorphisms and small insertions and deletions, a subset of which are present exclusively in the th mouse. a complete catalogue of th genomic variants will aid the identification of causative variants and genes underlying the diseases observed in th mice, which will maximize the relevancy of this model for human obesity and type diabetes. knowing the distribution of variants across the genome of the th mouse vs. other classic and wild-derived inbred strains may identify naturally occurring gene variant sets that can represent model genes for the complex human obesity and type diabetes. as a proof of principle, we integrated our genome variant data with genetic mapping data and identified a variant in cell death-inducing dffa-like effector c whose effect on protein function we characterized. methods description and origin of the th mouse strain the th mouse is originally derived from an outbred colony of the theiler original strain in which two male mice spontaneously became polyuric, glucosuric, hyperinsulinemic, and hyperglycemic. in male diabetic progeny and apparently normal female mice were imported into the jackson laboratory and an inbred strain was then established by selecting for male hyperglycemia by dr. jürgen naggert’s research group at tjl. in a sub-colony was initiated in our laboratory with breeding pairs from dr. naggert’s research colony and used in this study. after arrival at our laboratory, we interbred siblings for generations followed by one generation backcrossing and an additional generations of sibling interbreeding. since the generation prior to arrival at our laboratory remains unknown, we refer to the generation used for whole genome sequencing in this study as f? + f18n1f all animal studies were carried out with the approval of marshall university animal care and use committee. dna sequencing, read alignment, and bioinformatics high quality genomic dna was extracted from the liver of a male th mouse from our colony using a qiagen genomic-tip 100/g kit. genomic dna was sheared using a covaris s instrument to ~350 bp and used to construct a sequencing library using an illumina dna sample preparation kit according to the manufacturer’s recommended protocol. the library was quantified using thermofisher qubit fluorimetry and sized on an agilent bioanalyzer dna chip. the resulting whole genome library was amplified on a flow cell using an illumina cbot cluster station and then sequenced on an illumina hiseq in the marshall university genomics core facility. three sequencing runs were performed in order to obtain adequate depth of coverage. sequencing reads were aligned to the c57bl/6j reference genome using bowtie v.. our variant calling pipeline was based on that developed by wong et al. to call variants in the fvb/nj mouse strain. briefly, duplicate reads were removed with samtools v. using the “samtools rmdup” command. to improve quality of the variant calling, local realignment around insertions and deletions was performed using gatk v. by first running the “realignertargetcreator” command and then the “indelrealigner” command. snps and indels were called by generating a pileup using the “samtools pileup” command with options ueds, and piping the results to the “bcftools view” command with options “-p –vbcgn”. variants were filtered using the vcftools package version. in order to maintain maximal consistency with the mouse genome project, we used the same options as in wong et al.. we identified variants in the th strain that did not occur in any of the mouse strains published in the mgp, which we term “private” variants, following keane et al.. to do this, we generated a list of all genomic locations of th variants in the form of a bed file, and then performed variant calling on the bam files for the mgp mouse strains at those locations, using samtools’ “–l” option along with the same parameters as were used to call th variants. th snps qualified as private if no mgp strain had the same snp at the same location, where at least of the strains had a call quality at least and read depth at least at that location. th indels qualified as private if no mgp strain had a variant at the same genomic location, with the same criteria for call quality and read depth being applied. we then added functional consequence annotation, including “sorting intolerant from tolerant” prediction of protein function changes of coding variants, using a local installation of ensembl variant effect predictor version. output from vep includes classification of each variant using a set of one or more sequence ontology terms. variants are classified multiple times if they lie within a region intersecting multiple known transcripts. we tabulated the number of variants according to their collection of so terms, collecting into a single “multiple classification sets” group all variants that had multiple, distinct sets of so terms due to their presence in multiple transcripts. since this latter group is large, and may include potentially interesting variants, we identified a set of ten so terms we call “potentially pathogenic”: frameshift_variant, inframe_deletion, inframe_insertion, missense_variant, stop_gained, stop_lost, initiator_codon_variant, splice_region_variant, splice_acceptor_variant, and splice_donor_variant. we tabulated the number of variants in the “multiple classification sets” that classified with each of these so terms for one or more transcripts. the same classification was performed on the private variants. for missense variants, we additionally used protein variation effect analyzer v to provide further prediction of the functional effects of protein changes. in order to uniquely associate a representative so term with each variant, we chose a single representative so term for each distinct set of so terms identified for a given variant in a given transcript, as shown in additional file 2: table s and additional file 3: table s for example, snps were classified with the two so terms “intron_variant” and “splice_region_variant”: for these snps we chose “splice_region_variant” as the representative term. using this process, variants occurring in multiple transcripts that had multiple, distinct sets of so terms associated with them, resulted in having multiple representative so terms. for such variants, we chose the “most pathogenic” representative so term, ordering them in the following priority: “stop_gained”, “stop_lost”, “frameshift_variant”, “missense_variant”, “inframe_insertion”, “inframe_deletion”, “splice_acceptor_variant”, “splice_donor_variant”, “splice_region_variant”, “initiator_codon_variant”, “synonymous_variant”, “5_prime_utr_variant”, “3_prime_utr_variant”, “mature_mirna_variant”, “upstream_gene_variant”, “downstream_gene_variant”, “intron_variant”, “non_coding_transcript_variant”, “intergenic_variant”. in order to compare our variant sets to known associations between genetic variants and human traits for which th is a model, we first retrieved the genome wide association study catalog from the european bioinformatics institute, and filtered the catalog by the column “disease/trait” for each of the terms “obesity”, “diabetes”, and “metabolic”. for each filtered catalog, we extracted all human entrez geneids listed in the columns mapped gene, upstream_gene_id, and downstream_gene_id. using the ensembl interface to biomart, we converted these entrez geneids to ensembl human gene ids. additionally, we retrieved a list of genes from pigeyre et al. in which mutations have been previously shown to cause a monogenic obesity phenotype in humans, and found ensembl ids for these genes using the same tool. for all these human ensembl gene ids, we found ensembl mouse gene ids of orthologous genes, again using the ensembl interface to biomart. we then filtered our variant sets to identify potentially pathogenic variants associated with each of these orthologous gene sets. aligned reads were uploaded to the sequence read archive at the national center for biotechnology information and can be accessed via accession number srp cell culture, plasmids, transfections, and microscopy cos- cells were grown in dulbecco’s modified eagle’s medium with l-glutamine,  g/l glucose and sodium pyruvate supplemented with 10 % bovine serum and units/ml penicillin and 100 mg/ml streptomycin. the murine cidec cdna was procured from genscript. a green fluorescent protein fusion, pacgfp1-cidec was created by cloning the cidec cdna with 5′ hindiii and 3′ bamhi sites upstream and in-frame with the acgfp sequence in pacgfp1-n. the entire cidec coding region was included except for the stop codon. site-directed mutagenesis was used to introduce the missense polymorphism r46s. plasmids, pacgfp1-cidec and pacgfp1-cidec, were transfected into cos- cells cultured on 4-well chamber slides using lipofectamine ltx&plus following the manufacturer’s instruction. cos cells do not express endogenous cidec. after transfection, the cells were cultured for 48 h with and without 400 μm bsa-complexed oleic acid. cells were then washed with pbs, fixed by 4 % paraformaldehyde for 30 min, and permeabilized in  % saponin in pbs for 20 min. lipids were stained with 1 μg/ml nile red which partitions with neutral lipids; cells were washed with pbs. nuclei were labeled by placing coverslips onto slides with prolong® gold antifade mountant with dapi. coverslips were dried and cells viewed on a leica sp confocal microscope located in the marshall university imaging center. each experiment was performed in quadruplicate samples. association of full length cidec with lipid droplets in cos- cells was assessed using imagej software by performing a co-localization analysis of gfp florescence with nile red labeled lipid droplets in each z-section of a transfected cell; these results were then averaged to calculate the overall co-localization in a given cell. the data were from cell clusters in each condition from two independent experiments. quantitative data were presented as means ± sem. two-tailed student’s t-tests were performed on data. differences of p <  were considered significant.  identification of snps and indels the whole genome of the th mouse was sequenced to an average depth of ~ x coverage using data from three illumina paired-end read sequencing runs. the sequencing reads were mapped to the b mouse reference genome. using a samtools-based pipeline, we identified, snps and, indels genome-wide, relative to the reference strain c57bl/. the positional distribution of variants on each chromosome is shown in fig.  to assess the reliability of our data, pcr amplification and sanger sequencing were applied to homozygous snps to determine whether they agreed with next generation sequencing results. we found that all snps were consistent with the illumina sequencing data.fig. distribution of detected variants on the th genome. total number of variants, number of snps, and number of indels per megabase as a function of genomic position. these were calculated by sliding a one-megabase window across each chromosome in steps of one kilobase and totaling the number of variants in the window we observed a small percentage of heterozygous snps and indels in the th genome. possible explanations for the apparent presence of heterozygosity include errors in variant calling, true residual heterozygosity, and recent heterozygous mutations not yet fixed to homozygosity. we did not consider heterozygous snps and indels in our subsequent analyses. functional consequences of snps and indels we assigned putative functional consequences to the set of homozygous variants in th using variant effect predictor, which classified the variants using terms from the sequence ontology. variants which intersected multiple transcripts, and for which the set of so terms associated with the variant differs among those transcripts, were categorized as “multiple classification sets”. for each of ten so terms we designated “potentially pathogenic”, we counted the number of variants in the “multiple classification sets” category that classified under that so term in one or more transcripts. for each distinct collection of so terms identified for a variant, we chose a representative term. for variants with multiple classification sets this potentially resulted in multiple representative so terms: for these variants with multiple classifications we categorized the variant as the “most pathogenic” according to the prioritization defined in the methods. this resulted in a unique so term for each variant: the distributions of these so terms among snps and indels are shown in figs.  and respectively. the majority of snps were either intergenic or intronic. the next two largest snp groups were those which were located 5 kb upstream or downstream from a coding gene. a small number of snps were located within the protein coding regions of gene bodies. snp variants resulted in synonymous codon changes, non-synonymous codon changes, conversions of coding codons to stop codons and conversions of stop codons to coding codons.fig. classification of snps by sequence ontology term. the large pie chart shows the characterization of all tallyho/jng snps relative to the c57bl/ reference genome; the smaller chart shows the distribution of the subset of these we term “potentially pathogenic”, as described in the methods fig. classification of indels by sequence ontology term. the large pie chart shows the characterization of all tallyho /jng indels relative to the c57bl/ reference genome; the smaller chart shows the distribution of the subset of these we term “potentially pathogenic”, as described in the methods there were snps causing non-synonymous changes to one or more protein-coding transcripts in genes. we evaluated the effect of the amino acid substitutions resulting from the snps using sift and provean. these algorithms classify the changes as either deleterious or tolerated/neutral. sift classified  % of the non-synonymous substitutions in genes as deleterious. provean classified  % in genes as deleterious.  % of the missense variants in genes were classified as deleterious by both algorithms. as expected, the vast majority of th indels fell into noncoding groups. the total in-frame and frameshift indels were and respectively. proportions of snps and indels in each functional class are similar to those found in the fvb/nj genome.table comparison of distribution of variants in tallyho/jng to that in fvb/nj comparison to the human genome-wide association study catalog and to known mendelian obesity genes in order to interpret our data in the context of human disease, we collected human gene ids in which variants were associated with obesity, diabetes, or metabolic syndrome, from the ebi gwas catalog version.. these gene ids were mapped to orthologous mouse ensembl gene ids using the ensembl interface to biomart, generating sets of orthologous mouse genes. we additionally collected a list of known mendelian obesity genes in humans from pigeyre et al., and generated a set of mouse orthologous genes as aforementioned. we then filtered the potentially pathogenic th variants against these gene sets to provide lists of potentially pathogenic variants for each of these diseases. we found genes with potentially pathogenic variants in the gwas obesity gene set, in the gwas diabetes gene set, and in the gwas metabolic gene set. additionally, we found genes with potentially pathogenic variants orthologous to genes known to cause monogenic syndromic obesity, four orthologous to genes known to cause monogenic non-syndromic obesity, and three orthologous to genes known to cause monogenic non-syndromic lipodystrophy.table variants in genes linked to traits of interest by gwas or in mendelian obesity genes th private snps and indels we identified variants that were present in the th strain and absent from mgp strains by passing the bam files for each strain through our variant-calling pipeline at each location where we identified a th variant. a th snp was identified as “private” if it was not called as the same snp in any of the other strains, and a th indel was identified as “private” is no variant was identified at the same location in any of the other strains. additionally, the designation of “private” required a call quality at least and read depth at least in at least of the strains. in this analysis, we identified a total of private snps and private indels, which represented  % and  % of the total snps and indels, respectively. we classified private variants by so term using vep as described in the analysis for all variants above. of the private snps, were missense substitutions, four resulted in the gain of a stop codon, and one in the loss of a stop codon. among the private indels, were frameshift indels and were in-frame indels. for these private snps and indels, along with those associated by vep with splice regions and initiator codons, we counted the number of such variants occurring in each gene. there were private snps meeting one or more of these criteria in genes and private indels meeting one or more of these criteria in genes. for the private snps, we found that of the genes had one or more private snps determined by sift to be deleterious missense variants, had one or more private snps determined by provean to be deleterious, with genes having snps determined to be deleterious by both algorithms.fig. classification of private snps by sequence ontology term. the large pie chart shows the characterization of all tallyho/jng snps relative to the c57bl/ reference genome that are not present in previously sequenced strains; the smaller chart shows the distribution of the subset of these we term “potentially pathogenic”, as described in the methods fig. classification of private indels by sequence ontology term. the large pie chart shows the characterization of all tallyho/jng indels relative to the c57bl/ reference genome that are not present in previously sequenced strains; the smaller chart shows the distribution of the subset of these we term “potentially pathogenic”, as described in the methods in the analysis linking our variant lists with human gwas studies and human mendelian obesity genes described above, we determined the number of variants in orthologous genes which were private to the th mouse. characterization of the tabw obesity qtl interval on chromosome 6 tabw is a major obesity qtl identified in th mice, and confirmed by a congenic strain on the b background. using subcongenic analysis we have determined that the effect of tabw on obesity could be attributed to two adjacent loci, tabw2a and tabw2b. there are and protein coding genes cataloged in the ensembl mouse annotation database in the tabw2a and tabw2b intervals, respectively. a total of snps and indels were found in the tabw2a and tabw2b intervals, of which snps and indels were in protein-coding genes. among those, only snps in the tabw2a interval and snps in the tabw2b interval were classified as deleterious by both sift and provean. we then conducted a literature review of the biological function of the genes in the tabw interval with snps classified as deleterious by either algorithm, searching for potential link to obesity. from this search, the cidec gene, containing a snp identified as deleterious by both sift and provean, drew our attention. cidec is a lipid droplet protein that is involved in the regulation of cellular lipid droplet size and lipid storage during lipid metabolism in adipocytes. a loss of function mutation in cidec causes a familial partial lipodystrophy. further, weight loss via a low calorie diet was correlated with a reduced gene expression of cidec in adipose tissue in humans.table numbers of variants in the tabw locus tabw2a tabw2b gcfc2 81941369 cgg/tgg r/w 7/17 0 - ccdc142 htra2 mogs rtkn 1700003e16rik tet3 aak1 gfpt1 d6ertd527e chchd6 89467434 cgt/tgt r/c 5/8 - zfyve20 lmod3 ttll3 cidec 113433131 cgt/agt r/s 3/6 0 - irak2 rpl28-ps4 ret 118179286 acc/atc t/i 5/20 0 - cacna1c cecr5 a2m 121661523 acg/aag t/k 21/36 0 - 8430419l09rik atf7ip bc049715 136840191 atg/acg m/t 1/1 - art4 mgp ptpro igbp1b pik3c2g 139967890 agt/tgt s/c 16/16 - chr chromosome, a.a. amino acid, ref reference, th tallyho snps predicted to be deleterious by both sift and provean are shown in bold there was one nucleotide substitution in the cidec coding sequence, c > a. the substitution of c > a resulted in an amino acid difference of r46s between b and th strains. to examine the function of the cidec r46s polymorphism, we created murine plasmids acgfp1-cidec and acgfp1-cidec and transiently transfected them into cos- cells. cos- cells have been used as models for monitoring cidec function in prior studies because they lack endogenous cidec activity. it was apparent that acgfp1-cidec was localized to distinct lipid droplets stained with nile red. when we considered nile red lipid staining specifically associated with gfp fluorescence, cos- cells transfected with the s variant exhibited an increase in lipid accumulation compared to cells transfected with the wild type r variant in the media both with and without oleic acid. we speculate that the s variant may be hypermorphic in that it enhances the function of cidec in promoting lipid accumulation in lipid droplets.fig. representative images of lipid droplets in cos- cells transfected with acgfp1-cidec and acgfp1-cidec. a and c present cells cultured in media without oleic acid and b and d present cells cultured in media with oa. images of nuclei labeled with dapi, neutral lipid labeled with nile red and location of acgfp were merged. e quantification of lipid specifically associated with gfp in cells. using imagej software, the ratio of nile red/gfp signal was calculated from lipid droplet clusters each for r and s without oa and lipid droplet clusters for r and lipid droplet clusters for s with oa. data are presented as means ± sem. *p <  discussion landscape of the tallyho genome we found a total of, variants in the th genome, compared to the reference b genome. as expected, the large majority of variants occurred in non-protein coding regions of the genome. the number of snps was broadly consistent with that reported by keane et al. and wong et al. for laboratory strains, and substantially fewer than the four wild-derived strains sequenced in the keane et al. study. we found  % to  % more indels when compared to those laboratory strains, and again considerably fewer than the four wild-derived strains. the proportion of private snps we observed was higher than of the laboratory strains from keane et al., with the two exceptions being the two strains nod/shiltj and nzo/hiltj. we also showed a lower proportion of private snps in the th strain than discovered in fvb/nj. the proportion of private indels we observed in th was higher than in these studies. although the origins of the theiler original mouse are unknown, the generally higher proportion of private variants in th is consistent with the possibility that th has a unique ancestor. we also note that our sequencing coverage of x was higher than in these other studies, increasing our general power to detect variants. classification of variants we provided a comprehensive catalog of variants of the th mouse, relative to the b mouse, classified according to the effect of the variant on protein sequence. these effects were predicted by comparing the location of the variant on the reference genome to known genomic annotations provided by the ensembl gene database. while it is relatively straightforward to automate the classification of a large number of variants via genomic annotations on a coarse scale, providing detailed interpretations of the effect of some mutations requires greater inspection of the context of the mutation that cannot be readily automated and performed on a genome-wide scale. for example, the effect of intronic mutations is not well characterized or understood at present. our aims in this study include both providing a general overview of the landscape of the th mouse genome relative to the reference genome, and also providing sufficient detail of the nature of the million variants discovered. to this end, we used an automated process to classify variants into a large number of precisely defined categories, and then subsequently reduced the number of these categories, subjectively categorizing them into a smaller set of representative terms. the first categorization provides a fine-grained resource for searching for individual variants that may have phenotypic consequences, and the second provides a general overview of the landscape of the th genome. the sequence ontology provides a controlled vocabulary for describing features and annotations associated with a biological sequence, including mutations at the sequence level, along with a formalized description of the relationships between these terms. the variant effect predictor is a tool that predicts the effect of genomic variants by analyzing the variant in the context of annotations on the reference genomic sequence. for each variant and each known transcript containing that variant, vep outputs a subset of so terms associated with that variant. in many cases, this results in multiple so terms being associated with a single variant in the context of a single transcript. for example, we found an a to g single nucleotide polymorphism at genomic location, on chromosome this snp occurs in the second nucleotide of the first codon of the second exon of the apolipoprotein b receptor gene, apobr, mutating the gac codon to ggc. since this mutation results in a change in amino acid from aspartic acid to glycine, the snp is annotated with the so term “missense_variant”. since this variant also occurs within three bases of a splice site, it is also annotated with the so term “splice_region_variant”. in order to provide a complete, unbiased characterization of the types of variants discovered, we list all unique combinations of so terms for a single variant and the number of times those combinations occur. in order to provide a concise overview of the relative proportions of occurrences of the major types of variants, for each unique combination of so terms we selected a “representative” so term. these overviews are presented in figs.  and due to the presence of multiple transcripts for some genes, and due to the presence of overlapping genes, some individual variants are present in multiple transcripts. in this case, vep outputs a potentially different combination of so terms for each of the transcripts in which the variant occurs. when there are multiple, distinct, combinations for a given variant due to its presence in multiple transcripts, these variants are categorized in additional file 2: table s and additional file 3: table s as “multiple classification sets”. in order to uniquely classify such variants in the overview, we picked a representative so term for each set of so terms using the strategy shown in additional file 2: table s and additional file 3: table s and then chose the most pathogenic out of these representative so terms using the priority described in the methods. filtering variants by the human gwas catalog we employed a translational methodology to identify and prioritize candidate genes for complex disorders for which th is a model. we examined all the th variants for potential link to relevant human disease using the ebi gwas catalog and generated enriched candidate gene lists for obesity, diabetes and metabolic syndrome. we reviewed biological function for selected genes and identify genes we could connect to obesity and/or diabetes. glis is a transcription factor that plays an important role in pancreatic development and insulin gene expression in beta cells. sorbs is involved in insulin stimulated glucose transport in adipocytes. igf2bp is a rna binding protein, participating in posttranscriptional rna processing, i.e., rna splicing, stabilization, transport, and translation. inactivation of the igf2bp gene caused a resistance to diet-induced obesity in mice. semaphorin 5a is an axon regulator molecule and plays a role during neuronal and vascular development. collectively, our convergent approaches systematically integrate whole genome sequencing data and genetic information from gwas-derived findings and provide an opportunity to discover candidate genes for further functional validation. missense polymorphism of r46s in cidec between b and th mice among the genes with snps that were identified as deleterious by both sift and provean, were orthologous to mendelian lipodystrophy genes, and were contained within the tabw interval, we identified cidec, which is an important regulator of energy homeostasis directly involved in promoting the accumulation of triglyceride into intracellular lipid droplets. lipid droplets are spherical organelles found in many types of eukaryotic cells, including adipocytes, and are composed of a core of neutral lipids, such as sterol esters or triglycerides, surrounded by a monolayer of phospholipids, free cholesterol, and multiple specific proteins including cidec. in adipocytes, cellular energy is stored as triglycerides in lipid droplets, and in conditions of fatty acid excess, lipid droplets rapidly increase their volumes. the capacity of cidec in accumulating triglycerides into intracellular lipid droplets was demonstrated by transfection experiments using multiple cell types including c pre-adipocytes, 293t cells, and cos cells. for example, transfection of full-length murine cidec into cos- cells increased total cellular triglycerides by 50 %. we found that the th allele of cidec s variant allowed for more lipid accumulation than the b allele of cidec r variant in cos- cells. further functional validation of this missense polymorphism in vivo may provide an opportunity to understand the role of cidec in the context of obesity.  we have sequenced the whole genome of obese type diabetic th mice by next-generation sequencing and generated a complete catalog of variants classified by location relative to genes and predicted consequences for protein products. we filtered the list of variants to the tabw obesity qtl and identified a missense polymorphism in cidec whose protein function we characterized. furthermore, by comparing the variant catalog to the human gwas catalog and to known human mendelian obesity genes, we were able to identify a list of susceptibility candidate genes that could be used to dissect the components of polygenic diseases such as obesity and type diabetes. additional files additional file 1: table s mouse genome project strains. list of strains from the mouse genome project used in the determination of “private” variants. additional file 2: table s classification of th snps by sets of so terms associated with the snp. additional file 3: table s classification of th indels by sets of so terms associated with the indel. additional file 4: table s pcr primer sequences used for validation of selected variants. additional file 5: table s variants in orthologs of gwas-associated and mendelian genes. list of orthologous genes to human genes associated by gwas studies to obesity, diabetes, and metabolic syndrome, and to mendelian obesity genes, with variant type and number. additional file 6: table s private snp classification. classification of private th snps by sets of so terms associated with the snp. additional file 7: table s private indel classification. classification of private th indels by sets of so terms associated with the indel. additional file 8: table s genes with pathogenic private snps. list of genes with pathogenic private snps, with sift scores and so terms for the corresponding snp. additional file 9: table s genes with pathogenic private indels. list of genes with pathogenic private indels, with so terms for the corresponding indel. abbreviations dnadeoxyribonucleic acid ebieuropean bioinformatics institute gwasgenome wide association study indelinsertion and/or deletion mbasemegabase mgpmouse genome project ncbinational center for biotechnology information proveanprotein variation effect analyzer qtlquantitative trait locus siftsorting intolerant from tolerant snpsingle nucleotide polymorphism sosequence ontology srasequence read archive tabw2tallyho associated body weight 2 thtallyho/jng mouse strain tjlthe jackson laboratory vepvariant effect predictor goran boskovic deceased this work was supported in part by the nih/niddk-r01dk nih/ncrr p rr and nih/nigms p20gm which funds the idea wv-inbre program and supports the marshall university genomics core facility and in part by aha-0855300e. points of view in this document are those of the authors and do not necessarily represent the official position or views of the nih or the aha. we thank kristy d. dillon and taryn p. stewart for maintaining mouse colonies, and david neff in the marshall university imaging core facility for his assistance in confocal microscopy. availability of data and materials raw sequencing data from this study are deposited in the national center for biotechnology information sequencing read archive and are available via accession number srp authors’ contributions jd performed all data processing and bioinformatics, including alignment, variant calling, identification of private variants, and cross-referencing to the gwas catalog and mendelian obesity genes. gb and jf prepared next-generation sequencing libraries and performed the sequencing. jhk and jkp prepared genomic dna and conducted cell culture and transfection experiments and pcr. jhk, jd and dap designed the overall research project and drafted the manuscript, tables and figures. all authors except gb, who died unexpectedly during the course of the study, read and approved the final manuscript. competing interest the authors declare that they have no competing interests.
further, we filtered our list of variants to those occurring in an obesity quantitative trait locus, tabw identified in th mice and found a missense polymorphism in the cidec gene and characterized this variant’s effect on protein function. the tallyho/jng mouse is a polygenic model for obesity and type diabetes first described in the literature in the origin of the th strain is an outbred colony of the theiler original strain and mice derived from this source were selectively bred for male hyperglycemia establishing an inbred strain at the jackson laboratory. in order to identify potentially clinically-relevant genes, we intersected our list of snp and indel variants with human orthologous genes in which variants were associated in gwas studies with obesity, diabetes, and metabolic syndrome, and with genes previously shown to confer a monogenic obesity phenotype in humans, and found several candidate variants that could be functionally tested using th mice. th mice manifest many of the disease phenotypes observed in human obesity and type diabetes. we sequenced the whole genome of th mice maintained at marshall university to a depth of approximately x coverage using data from three next generation sequencing runs. we generated a complete catalog of variants in th mice using the data from whole genome sequencing. our findings will facilitate the identification of causal variants that underlie metabolic diseases in th mice and will enable identification of candidate susceptibility genes for complex human obesity and type diabetes. genome-wide, we found approximately million homozygous single nucleotide polymorphisms and million homozygous small insertions and deletions of which snps and indels were unique to the th strain compared to previously sequenced inbred mouse strains. whole genome sequencingobesitytype diabetesmouse modeltallyhoquantitative trait locihttp://dx.doi.org/ /100000062national institute of diabetes and digestive and kidney diseasesdk077202kim jung han http://dx.doi.org/http://dx.doi.org/ /100000097national center for research resourcesrr016477http://dx.doi.org/http://dx.doi.org/ /100000057national institute of general medical sciencesgm103434http://dx.doi.org/ /100000968american heart association0855300ekim jung han issue-copyright-statement© the author 2016
 the tallyho/jng mouse is a polygenic model for obesity and type diabetes first described in the literature in the origin of the th strain is an outbred colony of the theiler original strain and mice derived from this source were selectively bred for male hyperglycemia establishing an inbred strain at the jackson laboratory. th mice manifest many of the disease phenotypes observed in human obesity and type diabetes.  we sequenced the whole genome of th mice maintained at marshall university to a depth of approximately x coverage using data from three next generation sequencing runs. genome-wide, we found approximately million homozygous single nucleotide polymorphisms and million homozygous small insertions and deletions of which snps and indels were unique to the th strain compared to previously sequenced inbred mouse strains. in order to identify potentially clinically-relevant genes, we intersected our list of snp and indel variants with human orthologous genes in which variants were associated in gwas studies with obesity, diabetes, and metabolic syndrome, and with genes previously shown to confer a monogenic obesity phenotype in humans, and found several candidate variants that could be functionally tested using th mice. further, we filtered our list of variants to those occurring in an obesity quantitative trait locus, tabw identified in th mice and found a missense polymorphism in the cidec gene and characterized this variant’s effect on protein function.  we generated a complete catalog of variants in th mice using the data from whole genome sequencing. our findings will facilitate the identification of causal variants that underlie metabolic diseases in th mice and will enable identification of candidate susceptibility genes for complex human obesity and type diabetes. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords whole genome sequencingobesitytype diabetesmouse modeltallyhoquantitative trait locihttp://dx.doi.org/ /100000062national institute of diabetes and digestive and kidney diseasesdk077202kim jung han http://dx.doi.org/http://dx.doi.org/ /100000097national center for research resourcesrr016477http://dx.doi.org/http://dx.doi.org/ /100000057national institute of general medical sciencesgm103434http://dx.doi.org/ /100000968american heart association0855300ekim jung han issue-copyright-statement© the author 2016 