, Melissa Yssel, MB ChB, FC Path(SA) Chem 139, and Wendy M. Zakowicz, BS 79 Purpose: To achieve clinical validation of cutoff values for newborn screening by tandem mass spectrometry through a worldwide collaborative effort. Methods: Cumulative percentiles of amino acids and acylcarnitines in dried blood spots of approximately 25-30 million normal newborns and 10,742 deidentified true positive cases are compared to assign clinical significance, which is achieved when the median of a disorder range is, and usually markedly outside, either the 99th or the 1st percentile of the normal population. The cutoff target ranges of analytes and ratios are then defined as the interval between selected percentiles of the two populations. When overlaps occur, adjustments are made to maximize sensitivity and specificity taking all available factors into consideration.

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Mutations in the SLC25A15 gene, encoding the human inner mitochondrial membrane ornithine transporter, are thought to be responsible for hyperornithinemia-hyperammonemia-homocitrullinemia (HHH) syndrome, a rare autosomal recessive condition. HHH syndrome has been detected in several small, isolated communities in northern Saskatchewan (SK). To determine the incidence of HHH syndrome in these communities, a PCR method was set up to detect F188Δ, the common French-Canadian mutation. Neonatal blood spots collected from all newborns from the high risk area were genotyped for the F188Δ mutation for seven consecutive years. Using DNA analysis, we estimated that the heterozygote frequency for the mutant allele for HHH syndrome to be about 1 in 19 individuals, predicting one affected child with HHH syndrome for approximately every 1,500 individuals (1 in 1,550 live births; 1 child every 12 years) in this isolated population. The frequency for the mutant allele for HHH syndrome in this isolated community is probably the highest in the world for this rare disorder. We determined that ornithine levels, by tandem mass spectrometry, were not abnormal in newborns with F188Δ mutation, carriers and normals. Ornithine rises to abnormally high levels at some time after birth well past the time that the newborn screening blood spot is collected. The timing or the reasons for the delayed rise of ornithine in affected children with HHH syndrome have not been determined. Newborn screening for HHH Syndrome in this high risk population is only possible by detection of the mutant allele using DNA analysis.

Patients with medium-chain acyl-CoA dehydrogenase (MCAD) deficiency are unable to metabolize medium-chain fatty acids. Affected patients display a characteristic acylcarnitine profile when blood spots are collected after birth and analysed by tandem mass spectrometry. To determine the potential risk of metabolic decompensation in newborns with elevations of diagnostic metabolites (octanoylcarnitine>0.3, but <1 micromol/L), we investigated the relationship between octanoylcarnitine (C8) concentration in neonatal blood spots and the 985A>G MCAD genotype. Octanoylcarnitine values from 7140 newborns' blood spots were sorted. The highest C8 was approximately 0.7 micromol/L, which is below the range in classical MCAD deficiency. Samples with C8 levels above 0.25 micromol/L (group C) represented 1.4% of the total. Values between 0.05 and 0.25 micromol/L (group B) made up 87.8% of the total; 10.8% of the samples had C8 values less than 0.05 micromol/L (group A). One hundred samples from each group were selected at random and genomic DNA was amplified by PCR and analysed for the presence of the 985A>G mutation. The analysed samples from groups A and B were all homozygous normal. The 100 samples from group C contained 26 samples that were heterozygous for the 985A>G mutation. These findings indicated that the frequency distribution of heterozygotes is not random within this population. Group C was further divided into C1, the 26 heterozygotes, and C2, the remaining 74 newborns in group C. In group C1 only 2 (8%) were in the 'high-risk' group characterized by either low birth weight or requiring admission to the neonatal intensive care unit. In contrast, 28 (38%) from C2 had low birth weight or were in the neonatal intensive care unit. In our dataset, C8/C2 and C8/C12 ratios were also significantly elevated in both groups C1 and C2 compared to controls (group B). In contrast to what others have reported, the ratio of C8/C10 did not differentiate the group B controls from heterozygotes or other patients in metabolic distress (group C2), but were lower than those seen in classic MCAD or mild MCAD deficiency.