Quantitation of gamma-hydroxybutyric acid in dried blood spots: Feasibility assessment for newborn screening of succinic semialdehyde dehydrogenase (SSADH) deficiency

Abstract

ObjectiveSSADH deficiency, the most prevalent 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, in undiagnosed infants with otherwise routine childhood illnesses. We report pilot methodology on the feasibility of newborn screening for SSADH deficiency. MethodDried blood spot (DBS) cards from patients affected with SSADH deficiency were compared with 2831 archival DBS cards for gamma-hydroxybutyric acid content. Following extraction with methanol, GHB in DBS was separated and analyzed using ultra high-performance liquid chromatography tandem mass spectrometry. ResultsMethodology was validated to meet satisfactory accuracy and reproducibility criteria, including intra-day and inter-day validation. Archival refrigerated dried blood spot samples of babies, infants and children (N=2831) were screened for GHB, yielding a mean+/−S.D. of 8±5nM (99.9%-tile 63nM) (Min 0.0 Max 78nM). The measured mean and median concentrations in blood spots derived from seven SSADH deficient patients were 1182nM and 699nM respectively (Min 124, Max 4851nM). ConclusionsGHB concentration in all 2831 dried blood spot cards was well below the lowest concentration of affected children. These data provide proof-of-principle for screening methodology to detect SSADH deficiency with applicability to newborn screening and earlier diagnosis.