DNA carrier testing and newborn screening for maple syrup urine disease in Old Order Mennonite communities

Abstract

Maple syrup urine disease (MSUD) is an inherited metabolic disorder caused by mutations in the branched chain alpha-keto acid dehydrogenase complex. Worldwide incidence of MSUD is 1:225,000 live births. However, within Old Order Mennonite communities, the incidence is 1:150 live births and results from a common tyrosine to asparagine substitution (Y438N) in the E1alpha subunit of branched chain alpha-keto acid dehydrogenase. We developed a new DNA diagnostic assay utilizing TaqMan technology and compared its efficacy, sensitivity, and duration with an existing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Carrier testing was performed by both TaqMan technology and PCR-RFLP on DNA isolated from buccal swabs of 160 individuals as well as from buccal swabs and blood spots of nine at-risk newborns; assay time, sensitivity, and reliability were also evaluated. The TaqMan assay, like the PCR-RFLP assay, accurately determined Y438N E1alpha allele status. However, the TaqMan assay appeared (1) more sensitive than the PCR-RFLP assay, requiring 10-fold less DNA (10 ng) to reliably determine genotype status and (2) faster, reducing the assay time required for diagnosis from approximately 12 to 5 h. TaqMan technology allowed more rapid DNA diagnoses of MSUD in the neonate, thereby reducing the likelihood of neurological impairment while enhancing health and prognosis for affected infants.

FIG. 1.

Representative images of polymerase chain reaction–restriction fragment length polymorphism (A) and TaqMan (B) assays. (A) 4% agarose gel demonstrating the presence of a 170 bp band (Y438N allele) and/or a 147 bp band (normal allele). The presence of both bands indicates a carrier (Y438N/+). “NTC” indicates a no DNA control, and “uncut” indicates the 186 bp polymerase chain reaction product before ScaI restriction endonuclease digestion. (B) Normal:Y438N ratios were used to determine allele status. Normal or Y438N alleles had ratios of at least 2:1, whereas carriers had allele ratios of ∼1:1. Standards were harvested from commercially available cell lines (ATCC; Normal Standard: GM00651; Y438N/+ Standard: GM00650; Y438N Standard: GM01099).