Identification of galactose-1-phosphate uridyl transferase gene common mutations in dried blood spots.

BACKGROUND: The California newborn screening program uses newborns' dried blood spots (DBS) to screen for more than 45 genetic disorders. Deficiency of galactose-1-phosphate uridyl transferase (GALT) is one of the metabolic genetic disorders screened using newborn DBS. During follow-up tests, common mutations of the GALT gene have been identified using whole blood samples. To avoid the stress of drawing an additional blood sample from newborns who are identified as presumptive positive for galactosemia, we developed a method to test common mutations in the GALT gene using blood spots. METHODS: This method involves DNA extraction from DBS, followed by polymerase chain reaction (PCR), and single nucleotide extension (SNE). SNE products were detected by capillary electrophoresis. RESULTS: In a double-blind study, GALT gene common mutations/variants: IVS2-2A>G, p.S135L, p.T138M, p.Q188R, p.L195P, p.Y209C, p.L218L, p.K285N, and p.N314D were detected in seventy-three DBS which had previously been screened and confirmed as positive in the California Newborn Screening Program. Mutations found using blood spots gave 100% concordance with mutations from previously genotyped whole blood samples. CONCLUSIONS: This blood spot method decreases the genomic test turnaround time of GALT screened positive patients and potentially reduces emotional stress on families required to provide an additional blood draw.

An automated method on analysis of blood steroids using liquid chromatography tandem mass spectrometry: application to population screening for congenital adrenal hyperplasia in newborns.

BACKGROUND: Newborn screening for congenital adrenal hyperplasia (CAH) is commonly accomplished by measurement of 17-α-hydroxyprogestrone (17-OHP) using enzyme immunoassay (EIA). EIA contributes a significant number of false positives. Therefore, second-tier steroid profile by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is warranted. METHODS: Dried blood spots (DBS) were extracted with a mixture of methanol and water containing the deuterium labeled internal standards of d(8)-17-OHP, d(7)-androstenedione, and d(4)-cortisol. The final extracts were analyzed for 17-OHP, androstenedione and cortisol by LC-MS/MS in the multiple reaction monitoring (MRM) mode. RESULTS: Mean recoveries of the target analytes, 17-OHP, androstenedione and cortisol, were between 97 and 115% with an average intra- and inter-assay CVs ranging from 3.9-9.9% to 3.6-10.1%, respectively. The high efficiency of this method enabled us to test 11,598 specimens, identified as indeterminate by EIA in ~6 years; resulting in 809 presumptive positives reducing the false positives rate by 93%. CONCLUSIONS: The three steroid profile provided better screening outcomes of CAH than 17-OHP concentration alone. Our sample preparation allowed high throughput using common laboratory chemicals. Using three internal standards significantly improved method precision and accuracy. The reduction in false positives significantly reduces anxiety for newborns and their families.

Improved tandem mass spectrometry (MS/MS) derivatized method for the detection of tyrosinemia type I, amino acids and acylcarnitine disorders using a single extraction process.

BACKGROUND: Succinylacetone (SUAC), a specific marker for tyrosinemia type I (Tyr I) cannot be detected by the routine LC-MS/MS screening of amino acids (AA) and acylcarnitines (AC) in newborns. The current derivatized methods require double extraction of newborn dried blood spots (DBS); one for AA and AC and the second for SUAC from the blood spot left after the first extraction. We have developed a method in which AA, AC and SUAC are extracted in a single extraction resulting in significant reduction in labor and assay time. METHODS: The 3.2 mm DBS were extracted by incubating at 45 °C for 45 min with 100 µl of acetonitrile (ACN)-water-formic acid mixture containing hydrazine and stable-isotope labeled internal standards of AA, AC and SUAC. The extract was derivatized with n-butanolic-HCl and analyzed by LC-MS/MS. RESULTS: The average inter-assay CVs for, AA, AC and SUAC were 10.1, 10.8 and 7.1% respectively. The extraction of analytes with ACN-water mixture showed no significant difference in their recovery compared to commonly used solvent MeOH. The concentration of hydrazine had considerable impact on SUAC extraction. CONCLUSION: We developed a new MS/MS derivatized method to detect AA/AC/SUAC in a single extraction process for screening Tyr I along with disorders of AA and AC.

California's experience implementing a pilot newborn supplemental screening program using tandem mass spectrometry.

OBJECTIVE: In response to a California legislative mandate, a pilot tandem mass spectrometry (MS/MS) screening program was undertaken by the Genetic Disease Branch of the California Department of Health Services between January 2002 and June 2003. This article outlines the Genetic Disease Branch approach to implementing the MS/MS pilot program and the program evaluation strategies used. METHODS: Through the use of multiple data collection methods, we were able to describe hospital participation patterns, screening test uptake, screening test performance, follow-up services utilization, and provider and family satisfaction with the educational materials and follow-up services provided. RESULTS: During the 18-month pilot program, just more than one half of California's 755,698 newborns were offered MS/MS screening; among this group, 90% of parents chose to have their newborns screened. Fifty-one newborns were identified with MS/MS-detectable disorders, among 461 patients referred for follow-up testing (0.13% of the screened population). One disorder was diagnosed successfully for every 6939 newborns screened and for every 9 infants referred (excluding phenylketonuria). The overall California population prevalence of MS/MS-detectable disorders was 1 case per 6500 infants (excluding phenylketonuria). The positive predictive value for medium-chain acyl-CoA dehydrogenase deficiency was 86.7%, whereas the positive predictive value for short-chain acyl-CoA dehydrogenase deficiency was 21.6%. For a sample from Hawaii, 1 isovaleric aciduria case was detected among 6132 newborns. CONCLUSIONS: Evaluation of the California MS/MS screening pilot program demonstrated that this technology was effective in identifying additional metabolic disorders. The positive predictive value of screening was particularly good for medium-chain acyl-CoA dehydrogenase deficiency. Overall, patient referral rates were very acceptable. The utility of the program was also demonstrated by positive reviews from patients and providers.