Single Nucleotide Polymorphisms

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Single Nucleotide Polymorphisms

• Jennifer Lyon
• Eskind Biomedical Library
• May 1, 2009
• CRC Workshop Series

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Types of Genetic Variations

• Single Nucleotide Polymorphisms (SNP)
• Single base pair changes
• GTCATTCGATT
• GTCAGTCGATT
• Indels
• Small insertion/deletions
• CTT------GATC
• CTTACGGATC
• Small variable repeats microsatellites
• ACGACGACGACGACGACG (6 copies)
• ACGACGACGACGACGACGACG (7 copies)
• Variable Long tandem repeats (can be dozens to
  hundreds to thousands)
• Chromosomal Aberrations Translocations,
  Inversions, etc.

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Focusing on SNPs

• Types of SNPs
• SNP nomenclature
• Resources for SNPs
• Examples and Challenges in Finding SNPs
• http//learn.genetics.utah.edu/content/health/phar
  ma/snips/

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SNPs Types

• SNPs can be categorized in a number of ways, the
  most common are by location and function
  (relative to a gene)
• Intragenic SNPs are often categorized by function
  are they in a coding region, an intron, part of
  the mRNA, outside the mRNA but still in the gene
  locus (i.e., in the promoter)
• Extragenic SNPs may be considered simply
  genomic or might be labeled relative to the
  nearest gene, ie. 5 or 3 to a gene
• An extragenic SNP may affect regulatory regions
  important in gene expression or other DNA
  functions such as DNA replication.

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SNP Functional Categories

• coding nonsynonymous
• Missense, nonsense, frame shift
• coding synonymous
• Intronic
• splice site
• mRNA utr
• 5' utr or 3' utr
• (gene) locus region (5 or 3 to the gene)
• near gene usually means within 2000bp of gene
• genomic/extragenic (distant from any gene)

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Coding Nonsynonymous SNPs

• Missense change an aa

http//www.ncbi.nlm.nih.gov/Class/NAWBIS/Modules/V
ariation/powerpoint/variation_files/frame.html
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Coding Non-Synonymous SNPs

• Nonsense
• Change an aa to a stop codon
• Results in a shortened protein
• Frame Shift
• Are really single-base indels
• Drop or add one base and the triplet reading
  frame is thrown out of shift, altering all
  downstream aas and usually resulting in an
  earlier stop codon

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SNP Nomenclature

• The Human Genome Variation Society
  (http//www.hgvs.org/mutnomen/recs.html) has
  proposed some guidelines for SNP nomenclature,
  but at the moment, there is minimal consistency.
• Different sources will refer to the same SNP in
  different ways
• While dbSNP identifiers (rs12345678) are
  becoming common, they are not required of
  publishing authors and not used in all cases.

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SNPs at Base-Pair Level

• The base-pair change is given in various forms
• A/C T?G CgtT 432GgtC T73C
• The HGVS nomenclature recommendations
• "c." for a coding DNA sequence (like  c.76AgtT)
  "g." for a genomic sequence (like g.476AgtT) "m."
  for a mitochondrial sequence (like m.8993TgtC
• "r." for an RNA sequence (like r.76agtu)

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Position, position, position!

• The big issue with SNPs is identifying their
  location (numerically).
• Position can be specified
• Number location within a specific sequence
• Relative to another genetic landmark
• Start site for a coding region of a gene
• Start or end of an exon or intron
• Relative to a marker
• Published articles are not always clear on
  this!!!
• Different resources may use different
  landmarks/numbering
• Numbering is always relative to the chosen
  sequence

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Coding SNPs

• These are easier because they can be identified
  by the amino acid position rather than the
  base-pair position
• Most common nomenclature uses either 3-letter or
  single amino acid codes
• Asn332Asp OR A95V
• The HGVS recommendation is similar
• "p." for a protein sequence (like  p.Lys76Asn)
• Amino Acid (protein) coding sequence positions
  becoming more consistent, but are not always
  consistent

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Database of SNPs (dbSNP)

• dbSNP
• is the international central repository for both
  single base nucleotide substitutions and short
  deletion and insertion polymorphisms
• accepts data submissions from scientists
• is integrated with the NCBIs Entrez system

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dbSNP Content

• The SNP database has two major classes of
  content
• Submitted data, i.e., original observations of
  sequence variation Submitted SNPs (SS) with ss
  (ss 5586300)
• Computed/curated data Reference SNP Clusters
  (Ref SNP) with rs (rs 4986582)

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Reference SNP Clusters

• Ref SNP clusters are computer-generated and
  curated by NCBI staff
• Ref SNP Clusters define a non-redundant set of
  SNPs
• All individual SNPs submitted by a researcher are
  given a submitter SNP number (ss) and then
  redundant (repetitive) submitter SNPs are
  combined into a RefSNP cluster record, with a
  unique rs
• Ref SNP clusters may contain multiple submitted
  SNPs

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Searching dbSNP

• dbSNP is searched like any other Entrez db
• Specialized fields include

Field Tag Notes
Allele Allele Uses IUPAC codes for bases
Chromosomal Location CHRPOS Uses chromosomal base-pair locations
Contig Position ctpos Uses contig base-pair locations
Function Class Func Includes coding synonymous, missense, nonsense, intron, utr, etc.
SNP Class SNP_Class Includes snp, indel, mixed
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SNP Limits Page
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Creating a Complex Search

• Retrieve all synonymous coding reference SNPs for
  the human norepinephrine transporter gene
  (Slc6a2) from dbSNP
• Search Strategy
• humanorgn AND Slc6a2gene AND coding
  synonymous FUNC
• Note To use the gene (gene name) field, it is
  necessary to have the official gene name or gene
  symbol as per the Human Gene Nomenclature
  Committee. Entrez Gene can be used to find these.

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dbSNP Output Graphical Display
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dbSNP - Live

• Lets look at a dbSNP reference SNP page
• http//www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs374
  3788

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Finding SNPs - Challenges

• If rs is available start with it
• Not all rss have information in all databases
• Another database of interest is the Online
  Mendelian Inheritance in Man (OMIM)
• OMIM doesnt always provide rss even when there
  is one
• dbSNP records may link to OMIM or may not, even
  if the SNP is in an OMIM record

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Example 1

• rs1800888
• (CgtT) ? Ile164Thr in ADRB2 gene
• HGVS nomenclature
• NP_000015.1p.T164I
• To Find in OMIM
• Search with rs1800888 yield nothing
• Search with ADRB2gene find record
• Look at allelic variants .0003
  BETA-2-ADRENORECEPTOR AGONIST, REDUCED RESPONSE
  TO ADRB2, THR164ILE
• It is a match

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Example 2

• rs2740574
• A/G SNP located 5 to CYP3A4
• HGVS nomenclature
• NT_007933.14g.24616372CgtT
• To find in OMIM
• Search with rs2740574 yields nothing
• Search with gene name CYP3A4 find record
• Find list of allelic variants - .0001 CYP3A4
  PROMOTER POLYMORPHISM CYP3A4, a-g PROMOTER
• Compare info in dbSNP to info in OMIM (look at
  sequence)

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Other Databases

• OMIM NCBI
• HapMap - International HapMap Project
• ALFRED Allele Frequence Databases
• HGVbaseG2P - Human Genome Variation database of
  Genotype-to-Phenotype information
• PharmGKB Pharmacogenomics Knowledgebase
• F-SNP Functional SNPs