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Locus Reference Genomic (LRG) Sequences

Locus Reference Genomic (LRG) Sequences. Raymond Dalgleish Department of Genetics University of Leicester. Background. Descriptions of sequence variants should use HGVS nomenclature

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Locus Reference Genomic (LRG) Sequences

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  1. Locus Reference Genomic (LRG) Sequences Raymond DalgleishDepartment of GeneticsUniversity of Leicester

  2. Background • Descriptions of sequence variants should use HGVS nomenclature • Variants should be described with respect to a reference DNA sequence specified by an accession number and a versione.g. NM_000088.3:c.2362G>T • Mostly works well, but three key issues frequently cause problems for LSDB curators and for diagnostic laboratories

  3. Issue 1: Version not specified • The autosomal dominant RP10 form of retinitis pigmentosa is caused by variants in the IMPDH1 gene • Variants for this gene are described with respect to NM_000883.1, but the version is rarely mentioned in the literature • The current version (NM_000883.3) records a shorter mRNA & protein which could lead to confusion and delay

  4. Issue 2: Alternative splicing • ~93% of genes have alternatively spliced transcripts & may yield several proteins • The CDKN2A locus encodes the tumour suppressor proteins p16INK4a and p14ARF • The mRNAs for the two proteins share exon 2 in common but in different reading frames, due to different upstream exons • Separate RefSeq records for the mRNAs

  5. CDKN2A alternate splicing

  6. Issue 3: Legacy numbering (1) • The “sickle cell” variant of β-globin is due to the substitution of glutamic acid by valine at amino acid 6 • Determined by amino acid sequencing prior to completion of the genetic code • HGVS protein-level description is p.Glu7Val counting from the start codon

  7. Issue 3: Legacy numbering (2) • Type I & III collagen variants were originally numbered from the start of the Gly-X-Y triple-helical repeat region • Legacy and HGVS descriptions still run in parallel: e.g. Gly610Cys & p.Gly788Cys • The exons of these genes were originally numbered in a 3´ to 5´ direction

  8. Issue 3: Legacy numbering (3) • New exons are often discovered in genes long after their initial characterisation • This interferes with simple sequential numbering of exons from 5´ to 3´ • Non-simple numbering is well-established: • COL1A1: 33/34 • CFTR: 6a, 6b,14a, 14b, 17a, 17b • OPRM: O, X, Y • CDKN2A: 1B, 1A

  9. So what is the solution? • An ideal reference sequence would: • be stable over periods as long as 25 years • be free of version confusion • comprise an “idealised” genomic DNA sequence haplotype providing a practical working framework • contain comprehensive information about the transcripts and proteins encoded by the gene (including alternative numbering schemes) • be mapped to the current genome assembly

  10. Primary design decisions • LRGs will be a working representation of a gene with a permanent ID: i.e. no versions • Based on any existing RefSeqGene record • 5 kb upstream and 2 kb downstream • There can be more than one LRG for a given region of the genome • LRGs will have both fixed and updatable feature annotations

  11. Primary fixed annotations • Coding sequence coordinates • Transcripts essential to the reporting of sequence variants • The conceptual translated protein(s) • Non-coding transcripts

  12. Primary updatable annotations • Mapping to current genome assembly • Chromosome number • Any alternative IDs • Cross references to other reference sequences • “Legacy” exon and amino acid numbering systems • Links to LSDBs • Overlapping genes

  13. Variant reporting with LRGs • The calcitonin gene (CALCA) encodes the peptide hormones calcitonin and calcitonin gene related peptide (CGRP) by alternative splicing • A SNP in the first base of exon 4 affects the transcript (t2) and the resulting precursor protein (p2) for calcitonin • The variant can be reported at gene, mRNA and protein level with reference just to LRG_13 (CALCA)

  14. Progress • LRGs can be viewed at the LRG web site: http://www.lrg-sequence.org • The first 10 LRGs have been finalised: • COL1A1, COL1A2, COL3A1, CRTAP, ATP1A2, CACNA1A, SCN1A, PPIB, FKBP10, CALCA • Another 4 await final approval: • LEPRE1, CDKN2A, L1CAM, UBE3A • Requests have been received for around 100 others

  15. Other tools to view LRGs • Ensembl, NCBI Genome Workbench, NCBI Sequence Viewer will soon provide support for LRGs • NGRL Universal Browser displays LRGs with links through to LSDBs and dbSNP • Mutalyzer will be updated to parse LRGs to support their use in LOVD • Alamut will probably be the first commercial software support for LRGs

  16. How do I learn more? • Dalgleish et al., 2010, Genome Medicine, in press • LRG web site:http://www.lrg-sequence.org • LRG specification document:http://www.lrg-sequence.org/docs/LRG.pdf • The LRG XML schema is available for download • E-mail addresses: • Request help: help@lrg-sequence.org • Provide feedback: feedback@lrg-sequence.org • Request a new LRG: request@lrg-sequence.org

  17. Acknowledgements

  18. Coordination and funding • LRGs were devised by the GEN2PHEN project: http://www.gen2phen.org • The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement number 200754 — the GEN2PHEN project

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