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Serious complications of homocystinuria caused by cystathionine beta-synthase deficiency can be prevented by early intervention. We determined the prevalence of 6 specific mutations in 1133 newborn blood samples. Our results suggest that homocystinuria is more common than previously reported. Newborn screening for homocystinuria through mutation detection should be further considered.

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 beta-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 < or = 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 MALDI-TOF MS assay was 1152 genotypes within 20 min., Conclusions: This high-level multiplex method is able to genotype 12 polymorphisms involved in folate or Hcy metabolism. The method is rapid and reproducible and could facilitate large-scale studies of the genetic basis of hyperhomocysteinemia and associated pathologies.

Human NK cells are characterized by the expression of surface receptors of the killer cell Ig-like receptor (KIR) family, which are involved in the specific recognition of pathogenic target cells. Each NK cell expresses and maintains an individual subset of inhibitory and stimulatory KIR and in this way contributes to a diversified NK cell repertoire. To date, the molecular basis for generation of clonally distributed KIR expression patterns has been elusive. Here, analyses of DNA methylation patterns of KIR genes in NK cell lines as well as in NK cells, freshly isolated from peripheral blood, demonstrated that a small CpG island surrounding the transcriptional start site of each KIR gene is consistently demethylated in expressed KIR and methylated in unexpressed KIR. DNA-demethylating treatment resulted in a rapid and stable induction of transcription and cell surface expression of all formerly unexpressed KIR in NK cell lines, NK cell clones, and freshly isolated NK cells, but not in other cell types. In vitro methylation of KIR CpG islands repressed reporter gene expression in NK cells. We conclude that clonal patterns of KIR expression are mainly epigenetically determined and maintained through DNA methylation.