The frequency of short-chain acyl-CoA dehydrogenase gene variants in the US population and correlation with the C(4)-acylcarnitine concentration in newborn blood spots.

Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is a clinically heterogeneous disorder. The clinical phenotype varies from fatal metabolic decompensation in early life to subtle adult onset, some patients remain asymptomatic. Two mutations (511C>T; 625G>A) have been described in exons 5 and 6 of the SCAD gene. Although they alter the structural and catalytic properties of the SCAD protein, these variants are not true disease-causing mutations but confer disease susceptibility. Previous studies found these gene variants to be common in Europeans. We aimed to establish the frequency of these variants in the US population and to determine whether the presence of these variants correlates with elevated butyrylcarnitine (C(4)-acylcarnitine) concentrations in newborn blood spots. Based on the analysis of 694 samples, we found that the allele frequency of the 625G>A variant was significantly higher (22%) than that of the 511C>T variant (3%). These gene variants were detected in either homozygous or compound heterozygous form in 7% of the study population. Additionally, the frequency of the 625G>A allele in the Hispanic population (30%) was significantly higher than that of the African-American (9%) and Asian (13%) subpopulations. A previously unreported variant, IVS 5 (-10) C>T, was identified in three African-American newborns (0.3%). The C(4)-acylcarnitine concentration in blood spots was significantly higher in subjects homozygous for the 625A variant when compared to those homozygous for the wild type (p<0.0001). However, none of the observed genotypes was associated with a concentration of C(4)-acylcarnitine that would be consistent with a biochemical diagnosis of SCAD deficiency.

Improved specificity of newborn screening for congenital adrenal hyperplasia by second-tier steroid profiling using tandem mass spectrometry.

BACKGROUND: Newborn screening for congenital adrenal hyperplasia (CAH) involves measurement of 17alpha-hydroxyprogesterone (17-OHP), usually by immunoassay. Because this testing has been characterized by high false-positive rates, we developed a steroid profiling method that uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) to measure 17-OHP, androstenedione, and cortisol simultaneously in blood spots. METHODS: Whole blood was eluted from a 4.8-mm (3/16-inch) dried-blood spot by an aqueous solution containing the deuterium-labeled internal standard d(8)-17-OHP. 17-OHP, androstenedione, and cortisol were extracted into diethyl ether, which was subsequently evaporated and the residue dissolved in LC mobile phase. This extract was injected into a LC-MS/MS equipped with pneumatically assisted electrospray. The steroids were quantified in the selected-reaction monitoring mode by use of peak areas in reference to the stable-isotope-labeled internal standard. We analyzed 857 newborn blood spots, including 14 blood spots of confirmed CAH cases and 101 of false-positive cases by conventional screening. RESULTS: Intra- and interassay CVs for 17-OHP were 7.2-20% and 3.9-18%, respectively, at concentrations of 2, 30, and 50 microg/L. At a cutoff for 17-OHP of 12.5 microg/L and a cutoff of 3.75 for the sum of peak areas for 17-OHP and androstenedione divided by the peak area for cortisol, 86 of the 101 false-positive samples were within reference values by LC-MS/MS, whereas the 742 normal and 14 true-positive results obtained by conventional screening were correctly classified. CONCLUSION: Steroid profiling in blood spots can identify false-positive results obtained by conventional newborn screening for CAH.