Gaviglio, Amy

Abstract Context Single-tier newborn screening (NBS) for congenital adrenal hyperplasia (CAH) using 17-hydroxyprogesterone (17OHP) measured by fluoroimmunoassay (FIA) in samples collected at 24 to 48 hours produces a high false-positive rate (FPR). Second-tier steroid testing can reduce the FPR and has been widely implemented. Objective We investigated the accuracy of an alternative multitier CAH NBS protocol that incorporates molecular testing of the CYP21A2 gene and reduces the first-tier 17OHP cutoff to minimize missed cases. Methods We create a Minnesota-specific CYP21A2 pathogenic variants panel; developed a rapid, high-throughput multiplex, allele-specific-primer-extension assay; and performed a 1-year retrospective analysis of Minnesota NBS results comparing metrics between a conventional steroid-based 2-tier protocol and a molecular-based multitier NBS protocol, applied post hoc. Results CYP21A2 gene sequencing of 103 Minnesota families resulted in a Minnesota-specific panel of 21 pathogenic variants. The Centers for Disease Control and Prevention created a molecular assay with 100% accuracy and reproducibility. Two-tier steroid-based screening of 68 659 live births during 2015 resulted in 2 false negatives (FNs), 91 FPs, and 1 true positive (TP). A 3-tier protocol with a lower first-tier steroid cutoff, second-tier 21-variant CYP21A2 panel, and third-tier CYP21A2 sequencing would have resulted in 0 FNs, 52 FPs, and 3 TPs. Conclusion Incorporation of molecular testing could improve the accuracy of CAH NBS, although some distinct challenges of molecular testing may need to be considered before implementation by NBS programs.

Screened for by all state newborn screening (NBS) programs in the United States, mitochondrial acetoacetyl-coenzyme A thiolase (T2), or β-ketothiolase, deficiency is a rare autosomal recessive disorder that causes ketoacidosis and hypoglycemia/hyperglycemia. Outcomes vary from normal development to severe cognitive impairment or even death after an acute episode of ketoacidosis. The classical biochemical profile of T2 deficiency is a result of null mutations in both alleles of the ACAT1 gene and consists of persistently increased urinary excretion of ketones, characteristic organic acids, and tiglylglycine as well as abnormal blood or plasma acylcarnitine profiles in acute and stable conditions. Early diagnosis and aggressive management can prevent further episodes of ketoacidosis and lead to normal development. We report the cases of 3 children, all subsequently found to have mutations predicted to be associated with no residual T2 enzymatic activity, but only 1 was identified by NBS in Minnesota since 2001. To our knowledge, this is the first description of compound heterozygotes for null mutations associated with no enzymatic activity exhibiting normal urinary organic acid, blood, and plasma acylcarnitine profiles when clinically well, thereby explaining the false-negative NBS results. We suggest that T2 deficiency may be underrecognized, because the incidence of T2 deficiency in Minnesota, on the basis of these 3 cases, is 1 in 232 000, higher than the reported <1 in 1 million incidence. Our cases emphasize that T2 deficiency must be considered in patients who present with ketoacidosis disproportionately severe to the triggering illness despite normal NBS results or nonspecific biochemical findings in blood and urine during asymptomatic periods.

SummaryNewborns identified with profound biotinidase deficiency (BTD) by the Minnesota Newborn Screening Program (MN NBS) between 1 October 2004 and 30 May 2008 were all from new immigrant groups. Thirty‐three positive cases of BTD were identified out of 264 727 infants screened by the Wolf colorimetric system during the period of this study by MN NBS. Five cases of profound BTD (0.1 to <0.6 nmol/min per ml) and 26 cases of partial BTD (0.9 to 2.3 nmol/min per ml) were later confirmed through measurement of serum biotinidase activity. The incidence of combined partial and profound BTD of 1/8540 and that of profound BTD of 1/52 945 in Minnesota are unusually high in comparison with the reported worldwide numbers of 1/61 067 for combined BTD and 1/137 401 for profound BTD. Four out of the 5 cases of profound BTD ascertained in the MN NBS cohort were of Somali ethnic background, and the remaining case was of Asian (Pakistani/Indian) ethnic background. All four Somali patients have the P497S mutation, with one of the four being homozygous for the mutation. The three compound heterozygotes all have a novel mutation (P142T) and two of them have another change (Y428Y) that has never been described. Within the last two decades, Minnesota has become home to an estimated 40 000 Somali immigrants and their children (<1% of the total Minnesota population). New population demographics prompt careful analysis of case cohorts to identify specific groups at risk for rare inborn errors of metabolism.