title: Accurate single-nucleotide polymorphism allele assignment in trisomic or duplicated regions by using a single base-extension assay with MALDI-TOF mass spectrometry.
abstract: BACKGROUND
  The accurate assignment of alleles embedded within trisomic or duplicated regions is an essential prerequisite for assessing the combined effects of single-nucleotide polymorphisms (SNPs) and genomic copy number. Such an integrated analysis is challenging because heterozygotes for such a SNP may be one of 2 genotypes-AAB or ABB. Established methods for SNP genotyping, however, can have difficulty discriminating between the 2 heterozygous trisomic genotypes. We developed a method for assigning heterozygous trisomic genotypes that uses the ratio of the height of the 2 allele peaks obtained by mass spectrometry after a single-base extension assay.
  
  
  METHODS
  Eighteen COL6A2 (collagen, type VI, alpha 2) SNPs were analyzed in euploid and trisomic individuals by means of a multiplexed single-base extension assay that generated allele-specific oligonucleotides of differing M(r) values for detection by MALDI-TOF mass spectrometry. Reference data (mean and SD) for the allele peak height ratios were determined from heterozygous euploid samples. The heterozygous trisomic genotypes were assigned by calculating the z score for each trisomic allele peak height ratio and by considering the sign (+/-) of the z score.
  
  
  RESULTS
  Heterozygous trisomic genotypes were assigned in 96.1% (range, 89.9%-100%) of the samples for each SNP analyzed. The genotypes obtained were reproduced in 95 (97.5%) of 97 loci retested in a second assay. Subsequently, the origin of nondisjunction was determined in 108 (82%) of 132 family trios with a Down syndrome child.
  
  
  CONCLUSIONS
  This approach enabled reliable genotyping of heterozygous trisomic samples and the determination of the origin of nondisjunction in Down syndrome family trios.
authors.affiliation: Department of Genomics of Common Disease, School of Public Health, Imperial College London, London, UK.
authors.forename: Anne L
authors.initials: AL
authors.lastname: Trewick
authors.forename: Julia S El-Sayed
authors.initials: JS
authors.lastname: Moustafa
authors.forename: Adam J
authors.initials: AJ
authors.lastname: de Smith
authors.forename: Philippe
authors.initials: P
authors.lastname: Froguel
authors.forename: Gottfried
authors.initials: G
authors.lastname: Greve
authors.forename: Pål R
authors.initials: PR
authors.lastname: Njølstad
authors.forename: Lachlan J M
authors.initials: LJ
authors.lastname: Coin
authors.forename: Alexandra I F
authors.initials: AI
authors.lastname: Blakemore
mesh_terms: D000483:Alleles; D024142:Collagen Type VI / Q000235:genetics*; D004314:Down Syndrome / Q000235:genetics*; D005260:Female; D018628:Gene Dosage; D020440:Gene Duplication; D005838:Genotype; D006579:Heterozygote; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D024142:Collagen Type VI