High-Level Multiplex Genotyping of Polymorphisms Involved in Folate or Homocysteine Metabolism by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Abstract

Background: Increased plasma total homocysteine (tHcy), a risk factor for cardiovascular disease, is related to genetic, environmental, and nutritional factors, in particular folate status. Future large epidemiologic studies of the genetic basis of hyperhomocysteinemia will require high-throughput assays for polymorphisms of genes related to folate and Hcy metabolism. Method: We developed a high-level multiplex genotyping method based on matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the detection of 12 polymorphisms in 8 genes involved in folate or Hcy metabolism. The assay includes methylenetetrahydrofolate reductase (MTHFR) 677C>T and 1298A>C, methionine synthase (MTR) 2756A>G, methionine synthase reductase (MTRR) 66A>G, cystathionine ␤-synthase (CBS) 844ins68 and 699C>T, transcobalamin II (TCII) 776C>G and 67A>G, reduced folate carrier-1 (RFC1) 80G>A, paraoxonase-1 (PON1) 575A>G and 163T>A, and betaine homocysteine methyltransferase (BHMT) 742G>A. Results: The failure rate of the assay was <1.7% and was attributable to unsuccessful DNA purification, nanoliter dispensing, and spectrum calibration. Most errors were related to identification of heterozygotes as homozygotes. The mean error rate was 0.26%, and error rates differed for the various single-nucleotide polymorphisms. Identification of CBS 844ins68 was carried out by a semiquantitative approach. The throughput of the

Novel Hairpin-Shaped Primer Assay To Study the Association of the −44 Single-Nucleotide Polymorphism of theDEFB1Gene with Early-Onset Periodontal Disease

“…Most of the methods available for SNP detection, such as techniques based on mass spectrometry (20), high-pressure liquid chromatography (23), microarray analysis (6), Taqman probes (16), or molecular beacons (25), are generally costly, time consuming, and/or difficult to automate, hindering their implementation in most laboratories.…”

Parallel minisequencing followed by multiplex matrix-assisted laser desorption/ionization mass spectrometry assay for β-thalassemia mutations

“…However, in the more restricted Taiwanese population, only eight point mutations account for a great majority (98%) of bthalassemia cases. Although multiplex minisequencing followed by multiplex analysis of sequencing products is a feasible genotyping approach for some genes (Meyer et al 2004;Ross et al 1998), technical difficulties have limited the general use of this method (Tost and Gut 2002). Because extension efficiency and optimum primer annealing temperature vary with the length and sequence of each primer (Syvanen 1999;Wang et al 2003), it is difficult to optimize the multiplex minisequencing reaction (Butler et al 2001).…”

Hyperhomocysteinemia in developing age and nutritional aspects of folates: an early cardiovascular risk factor

“…Alcuni all'inizio della terapia possono non rispondere per la grave deplezione di folati; pertanto un paziente non dovrebbe essere considerato non 6 Diuretici Anticonvulsivanti, carbamazepina, isoniazide e fentoina: interferiscono con i folati FARMACI CHE RIDUCONO L'OMOCISTEINA N-acetilcisteina, penicillamina (analogo della cisteina) * Recentemente è stata messo a punto una metodica, molto rapida, capace di tipizzare il polimorfismo di 12 genotipi coinvolti nel metabolismo dei folati e dell'omocisteina e precisamente: MTHFR C677T ed A1298C, methionine synthase (MTR) A2756G, methionine synthase reductase (MTRR) A66G, cystathionine beta-synthase (CBS) 844ins68 e C699T, transcobalamina II (TCII) C776G e A67G, reduced folate carrier-1 (RFC1) G80A, paraoxonase-1 (PON1) A575G e T163A, betaine homocysteine methyltransferase (BHMT) G742A. La metodica può facilitare studi su larga scala circa le basi genetiche dell'omocisteinemia e delle patologie associate [12]. responsivo alla vitamina B6 finché non sia stato aggiunto acido folico (5-15 mg/die) allo schema di trattamento.…”

“…Inoltre recentemente sono state individuate varie mutazioni fra cui la mutazione C677T della 5,10-metilentetraidrofolatoreduttasi (MTHFR), associata a termolabilità dell'enzima ed attività ridotta all'incirca del 50%, con prevalenza omozigote dal 5 al 20% nelle popolazioni caucasiche studiate [7][8][9][10][11][12] (tab. 1).…”

“…Questo è dovuto allo scarso consumo di vegetali crudi, non raro oggi nel-la II-III infanzia, ed è particolarmente frequente nei soggetti anziani. Tale carenza subclinica di folati da insufficiente apporto, talora si associa ad un alterato assorbimento per malattie infiammatorie croniche intestinali o interferenze farmacologiche (metotrexate, anticonvulsivanti) e, nei soggetti omozigoti per la mutazione MTHFR C677T con ridotta funzione enzimatica, per la maggiore richiesta di folati perché la via della rimetilazione funzioni adeguatamente [11,12,17]. Tale deficit può essere riportato alla normalità o incrementando l'apporto dietetico o con supplementazioni farmacologiche, con conseguente riduzione o normalizzazione dei livelli plasmatici di omocisteina.…”