Abstracts

Rationale: SSADH deficiency, an autosomal recessive disorder of GABA degradation, is characterized by elevated gamma-hydroxybutyric acid (GHB). Neurological outcomes may be improved with early intervention and anticipatory guidance. Morbidity has been compounded by complications, e.g. hypotonia and respiratory arrest, in undiagnosed infants with otherwise routine childhood illnesses. At least half of patients develop epilepsy, typically as myoclonic or generalized tonic-clonic seizures. SUDEP has been reported in one undiagnosed patient, identified from post-mortem analysis following identification of an affected living sibling. We report methodology used to perform newborn screening, which will enable widespread and early detection. Methods: Dried blood spots in affected patients were compared with controls. In this method, three 1/8' dried blood spot punches are extracted with 200 µL methanol containing 500 ng/mL d6-GHB deuterium-labeled internal standard. The extract is dried, reconstituted, and analyzed via ultra-performance liquid-chromatography tandem mass spectrometry. GHB is completely separated from isomeric and isobaric interferences using a step gradient. Multiple selected reaction monitoring for d6GHB is m/z 109->90 and for GHB 103->85. The same fragment is present in the spectra of the α-hydroxy and β-hydroxy isomers. Therefore major transitions for α-hydroxyisobutyric, α-hydroxybutyric, β-hydroxyisobutyric, and β-hydroxybutyric acids are monitored to verify separation from GHB. Results: The above method was validated as meeting satisfactory accuracy and reproducibility criteria, including intra-day precision and inter-day validation. Of 1,700 archival dried blood spots screened, GHB mean +/- S.D. was 8.2 + 5.1 nM (99%-tile 63 nM). The measured concentrations in blood spots of four SSADH deficiency patients were 124, 271, 609 and 715 nM. Conclusions: GHB concentrations in all 1,700 dried blood spot cards were well below the lowest concentration of affected children. This indicates that the screening methodology for SSADH deficiency is valid, with applicability to newborn screening and enabling earlier diagnosis. Children presenting with epilepsy and metabolic disease would benefit from newborn screening for gamma-hydroxybutyric aciduria.