Sequencing of Idd regions in the NOD mouse genome

The Sanger Institute is identifying genetic variations that may be associated with type 1 diabetes (T1D) by sequencing regions of the non-obese diabetic (NOD) mouse genome known to be associated with T1D and comparing them with the same areas of a diabetes-resistant strain of mouse.

The Wellcome Trust Sanger Institute has undertaken the genome sequencing of regions of the NOD mouse relevant to T1D, also known as insulin-dependent diabetes (Idd). Comparing the sequences of Idd candidate regions between the diabetes-sensitive NOD mouse and the diabetes-resistant C57BL/6J reference mouse will allow identification of genomic variations putatively associated with diabetes in mice and, by extension, in humans.

It is hoped that this research will provide a better understanding of potential immunogenomic loci responsible for the initiation and progression of autoimmune destruction of insulin-producing β-cells, eventually paving the way to potential targets in therapy. For more information on the project and the data, please see the publications tab or contact Charles Steward, project lead.

[The Jackson Laboratory]

Background

Idd regions currently annotated by HAVANA.

Idd regions currently annotated by HAVANA. [Genome Research Limited]

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T1D is a polygenic autoimmune disease that is characterised by hyperglycaemia and is fatal if not treated. It results from the progressive autoimmune T cell-mediated destruction of the insulin-producing pancreatic β-cells of the islets of Langerhans. Disease frequency is attributable to the interaction of the environment on alleles at numerous loci in the genome, with nearly 50 loci known to be involved. T1D is typically associated with specific allelic variants of the Major Histocompatability Complex (MHC) class I and class II genes within the MHC, a region of the genome that is critical for mounting immune and autoimmune responses. To date however, the MHC is the only locus that has been found to be essential for the manifestation of this disease.

The NOD mouse spontaneously develops T1D and is a model organism since it shares multiple characteristics with the human disease. Such characteristics include genetic polymorphisms that affect shared pathways, common antigenic targets, and the expression of class II MHC molecules displaying related peptides. The NOD mouse therefore represents a valuable tool for studying the genetics of T1D and for evaluating therapeutic interventions.

Funding

The NOD bacterial artificial chromosome (BAC) sequencing was funded by Immune Tolerance Network (ITN) Contract AI 15416, which was sponsored by the National Institute of Allergy and Infectious Diseases (NIAID), the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), and the Juvenile Diabetes Research Foundation International (JDRF).

Collaborations

We are working closely with the following organisations:

BAC libraries

Two NOD mouse BAC libraries were constructed and the BAC ends sequenced. Clones from the DIL NOD BAC library constructed by RIKEN Genomic Sciences Centre (Japan) in conjunction with the Diabetes and Inflammation Laboratory (DIL) (University of Cambridge) from the NOD/MrkTac mouse strain are designated DIL. Clones from the CHORI-29 NOD BAC library constructed by Pieter de Jong (Children's Hospital, Oakland, California, USA) from the NOD/ShiLtJ mouse strain are designated CHORI-29.

All NOD mouse BAC end-sequences have been submitted to the International Nucleotide Sequence Database Consortium (INSDC), deposited in the NCBI trace archive. We have generated a clone map from these two libraries by mapping the BAC end-sequences to the latest assembly of the C57BL/6J mouse reference genome sequence. These BAC end-sequence alignments can then be visualized in the Ensembl mouse genome browser where the alignments of both NOD BAC libraries can be accessed through the Distributed Annotation System (DAS).

The Mouse Genomes Project has used the Illumina platform to sequence the entire NOD/ShiLtJ genome and this should help to position unaligned BAC end-sequences to novel non-reference regions of the NOD genome. Further information about the BAC end-sequences, such as their alignment, variation data and Ensembl gene coverage, can be obtained from the NOD mouse ftp site.

NOD mouse libraries

Library construction, sequencing and mapping details for both NOD mouse BAC libraries.
Library name DIL NOD CHORI-29 NOD
Strain name NOD/MrkTac NOD/ShiLtJ
Source Female liver Male kidney
Vector pBACe3.6 pTARBAC2.1
Originator RIKEN Genomic Sciences Centre, Japan Children's Hospital Oakland, California, USA
Contact Dr. Jayne Danska BACPAC resources
Total number of BAC clones 196,032 110,976
Passed BAC clones 150,878 75,046
BAC clones mapped successfully 125,266 62,162
Passed BAC end-sequences 332,535 170,159
Sanger clone prefix bQ bCN
Average BAC insert size bp 149,809 205,413
Accession numbers of BAC end-sequences FR000001-FR332535 FR332536-FR502694

Sequence, annotation & analysis

Targeted sequencing

In conjunction with external collaborators studying the genetics of NOD mice, clones covering defined Idd candidate regions were selected for whole BAC sequencing from either of the two NOD mouse libraries using the BAC end-sequence alignments in Ensembl. Sequencing was then carried out using T7 and SP6 primers on the vector, and big dye terminator chemistry. In parallel, the quality of the corresponding sequence in the C57BL/6J reference mouse has been checked. Clones from the DIL NOD BAC library constructed by RIKEN Genomic Sciences Centre (Japan) in conjunction with the Diabetes and Inflammation Laboratory (DIL) (University of Cambridge) from the NOD/MrkTac mouse strain are designated DIL. Clones from the CHORI-29 NOD BAC library constructed by Pieter de Jong (Children's Hospital, Oakland, California, USA) from the NOD/ShiLtJ mouse strain are designated CHORI-29.

Finished clones from the targeted Idd candidate regions are displayed in the NOD clone sequence section of the website, where they can be downloaded either as individual clone sequences or larger contigs that make up the accession golden path (AGP). To access all the sequence for a specific region, select the Idd region from the relevant chromosome dropdown menu and then click on "Show AGP". Clicking to the right of the vertical green bar will download the complete sequence for a contig. The importance and utility of these high quality finished sequences is demonstrated further by the essential role that the NOD/ShiLtJ strain derived CHORI-29 NOD BACs played in calibrating the variation calling software for the Mouse Genomes Project. All sequences are publicly available via the INSDC.

The approximate coordinates for the syntenic Idd regions in the Black 6 mouse, based upon available NOD sequence are shown below and are not necessarily exact. To access the NOD sequence click on the region.

Region Chromosome Strain Library Mouse GRCm38 Coordinates
Idd1 (MHC) 17 NOD/MrkTac DIL 17:33681276-37969522
Idd1 (MHC) 17 NOD/ShiLtJ CHORI-29 17:33681276-38548659
Idd3 3 NOD/MrkTac DIL 3:36492618-37600833
Idd4.1 11 NOD/MrkTac DIL 11:69704895-71153537
Idd4.2 11 NOD/MrkTac DIL 11:72734492-74404570
Idd4.2Q 11 NOD/ShiLtJ CHORI-29 11:86785996-90007691
Idd5.1_CHORI 1 NOD/ShiLtJ CHORI-29 1:60694705-61117038
Idd5.1 1 NOD/MrkTac DIL 1:60564732-63711641
Idd5.3 1 NOD/MrkTac DIL 1:65533102-69307244
Idd5.4 1 NOD/MrkTac DIL 1:130232728-130661594
Idd6.1+2 6 NOD/ShiLtJ CHORI-29 6:143550839-149565172
Idd6.AM 6 NOD/ShiLtJ CHORI-29 6:129593784-131241919
Idd9.1 4 NOD/MrkTac DIL 4:128371876-131853368
Idd9.1M 4 NOD/MrkTac DIL 4:134841437-135252443
Idd9.2 4 NOD/MrkTac DIL 4:146049976-149895141
Idd9.3 4 NOD/MrkTac DIL 4:149556939-151385803
Idd10 3 NOD/MrkTac DIL 3:99848826-101467080
Idd16.1 17 NOD/ShiLtJ CHORI-29 17:27302611-29220265
Idd18.1 3 NOD/MrkTac DIL 3:109144756-109930492
Idd18.2 3 NOD/MrkTac DIL 3:103489414-104054885

Manual annotation and analysis

Analysed and manually annotated sequences have been generated using in-house developed software in accordance with the manual annotation guidelines, and are available through the Vertebrate Genome Annotation browser Vega. Completed C57BL/6J annotation can also be viewed in the Vega genome browser alongside the NOD sequence. This allows comparison of the genomic sequence and genes in the candidate loci between diabetes resistant and diabetes sensitive strains, looking for example for SNPs, and is a useful way of identifying regions of difference between the two mouse strains. We have analysed the homologous C57BL/6J mouse and NOD mouse sequences and identified variation consequences. These data are available from the NOD mouse ftp site.

Publications

  • The Vertebrate Genome Annotation browser 10 years on.

    Harrow JL, Steward CA, Frankish A, Gilbert JG, Gonzalez JM, Loveland JE, Mudge J, Sheppard D, Thomas M, Trevanion S and Wilming LG

    Nucleic acids research 2014;42;Database issue;D771-9

  • Fine mapping of type 1 diabetes regions Idd9.1 and Idd9.2 reveals genetic complexity.

    Hamilton-Williams EE, Rainbow DB, Cheung J, Christensen M, Lyons PA, Peterson LB, Steward CA, Sherman LA and Wicker LS

    Mammalian genome : official journal of the International Mammalian Genome Society 2013;24;9-10;358-75

  • The non-obese diabetic mouse sequence, annotation and variation resource: an aid for investigating type 1 diabetes.

    Steward CA, Gonzalez JM, Trevanion S, Sheppard D, Kerry G, Gilbert JG, Wicker LS, Rogers J and Harrow JL

    Database : the journal of biological databases and curation 2013;2013;bat032

  • The B10 Idd9.3 locus mediates accumulation of functionally superior CD137(+) regulatory T cells in the nonobese diabetic type 1 diabetes model.

    Kachapati K, Adams DE, Wu Y, Steward CA, Rainbow DB, Wicker LS, Mittler RS and Ridgway WM

    Journal of immunology (Baltimore, Md. : 1950) 2012;189;10;5001-15

  • Mouse genomic variation and its effect on phenotypes and gene regulation.

    Keane TM, Goodstadt L, Danecek P, White MA, Wong K, Yalcin B, Heger A, Agam A, Slater G, Goodson M, Furlotte NA, Eskin E, Nellåker C, Whitley H, Cleak J, Janowitz D, Hernandez-Pliego P, Edwards A, Belgard TG, Oliver PL, McIntyre RE, Bhomra A, Nicod J, Gan X, Yuan W, van der Weyden L, Steward CA, Bala S, Stalker J, Mott R, Durbin R, Jackson IJ, Czechanski A, Guerra-Assunção JA, Donahue LR, Reinholdt LG, Payseur BA, Ponting CP, Birney E, Flint J and Adams DJ

    Nature 2011;477;7364;289-94

  • Evidence that Cd101 is an autoimmune diabetes gene in nonobese diabetic mice.

    Rainbow DB, Moule C, Fraser HI, Clark J, Howlett SK, Burren O, Christensen M, Moody V, Steward CA, Mohammed JP, Fusakio ME, Masteller EL, Finger EB, Houchins JP, Naf D, Koentgen F, Ridgway WM, Todd JA, Bluestone JA, Peterson LB, Mattner J and Wicker LS

    Journal of immunology (Baltimore, Md. : 1950) 2011;187;1;325-36

  • Nonobese diabetic congenic strain analysis of autoimmune diabetes reveals genetic complexity of the Idd18 locus and identifies Vav3 as a candidate gene.

    Fraser HI, Dendrou CA, Healy B, Rainbow DB, Howlett S, Smink LJ, Gregory S, Steward CA, Todd JA, Peterson LB and Wicker LS

    Journal of immunology (Baltimore, Md. : 1950) 2010;184;9;5075-84

  • Genome-wide end-sequenced BAC resources for the NOD/MrkTac() and NOD/ShiLtJ() mouse genomes.

    Steward CA, Humphray S, Plumb B, Jones MC, Quail MA, Rice S, Cox T, Davies R, Bonfield J, Keane TM, Nefedov M, de Jong PJ, Lyons P, Wicker L, Todd J, Hayashizaki Y, Gulban O, Danska J, Harrow J, Hubbard T, Rogers J and Adams DJ

    Genomics 2010;95;2;105-10

  • Ly49 cluster sequence analysis in a mouse model of diabetes: an expanded repertoire of activating receptors in the NOD genome.

    Belanger S, Tai LH, Anderson SK and Makrigiannis AP

    Genes and immunity 2008;9;6;509-21

  • Interleukin-2 gene variation impairs regulatory T cell function and causes autoimmunity.

    Yamanouchi J, Rainbow D, Serra P, Howlett S, Hunter K, Garner VE, Gonzalez-Munoz A, Clark J, Veijola R, Cubbon R, Chen SL, Rosa R, Cumiskey AM, Serreze DV, Gregory S, Rogers J, Lyons PA, Healy B, Smink LJ, Todd JA, Peterson LB, Wicker LS and Santamaria P

    Nature genetics 2007;39;3;329-37

  • Molecular genetic analysis of the Idd4 locus implicates the IFN response in type 1 diabetes susceptibility in nonobese diabetic mice.

    Ivakine EA, Gulban OM, Mortin-Toth SM, Wankiewicz E, Scott C, Spurrell D, Canty A and Danska JS

    Journal of immunology (Baltimore, Md. : 1950) 2006;176;5;2976-90

  • Natural genetic variants influencing type 1 diabetes in humans and in the NOD mouse.

    Wicker LS, Moule CL, Fraser H, Penha-Goncalves C, Rainbow D, Garner VE, Chamberlain G, Hunter K, Howlett S, Clark J, Gonzalez-Munoz A, Cumiskey AM, Tiffen P, Howson J, Healy B, Smink LJ, Kingsnorth A, Lyons PA, Gregory S, Rogers J, Todd JA and Peterson LB

    Novartis Foundation symposium 2005;267;57-65; discussion 65-75

  • Fine mapping, gene content, comparative sequencing, and expression analyses support Ctla4 and Nramp1 as candidates for Idd5.1 and Idd5.2 in the nonobese diabetic mouse.

    Wicker LS, Chamberlain G, Hunter K, Rainbow D, Howlett S, Tiffen P, Clark J, Gonzalez-Munoz A, Cumiskey AM, Rosa RL, Howson JM, Smink LJ, Kingsnorth A, Lyons PA, Gregory S, Rogers J, Todd JA and Peterson LB

    Journal of immunology (Baltimore, Md. : 1950) 2004;173;1;164-73

  • Identification of a structurally distinct CD101 molecule encoded in the 950-kb Idd10 region of NOD mice.

    Penha-Gonçalves C, Moule C, Smink LJ, Howson J, Gregory S, Rogers J, Lyons PA, Suttie JJ, Lord CJ, Peterson LB, Todd JA and Wicker LS

    Diabetes 2003;52;6;1551-6

* quick link - http://q.sanger.ac.uk/qlnwiwiq