Quality considerations and major pitfalls for high throughput DNA-based newborn screening for severe combined immunodeficiency and spinal muscular atrophy.

BACKGROUND: Many newborn screening programs worldwide have introduced screening for diseases using DNA extracted from dried blood spots (DBS). In Germany, DNA-based assays are currently used to screen for severe combined immunodeficiency (SCID), spinal muscular atrophy (SMA), and sickle cell disease (SCD). METHODS: This study analysed the impact of pre-analytic DNA carry-over in sample preparation on the outcome of DNA-based newborn screening for SCID and SMA and compared the efficacy of rapid extraction versus automated protocols. Additionally, the distribution of T cell receptor excision circles (TREC) on DBS cards, commonly used for routine newborn screening, was determined. RESULTS: Contaminations from the punching procedure were detected in the SCID and SMA assays in all experimental setups tested. However, a careful evaluation of a cut-off allowed for a clear separation of true positive polymerase chain reaction (PCR) amplifications. Our rapid in-house extraction protocol produced similar amounts compared to automated commercial systems. Therefore, it can be used for reliable DNA-based screening. Additionally, the amount of extracted DNA significantly differs depending on the location of punching within a DBS. CONCLUSIONS: Newborn screening for SMA and SCID can be performed reliably. It is crucial to ensure that affected newborns are not overlooked. Therefore a carefully consideration of potential contaminating factors and the definition of appropriate cut-offs to minimise the risk of false results are of special concern. It is also important to note that the location of punching plays a pivotal role, and therefore an exact quantification of TREC numbers per µl may not be reliable and should therefore be avoided.

Neurological outcome in long-chain hydroxy fatty acid oxidation disorders.

OBJECTIVE: This study aims to elucidate the long-term benefit of newborn screening (NBS) for individuals with long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein (MTP) deficiency, inherited metabolic diseases included in NBS programs worldwide. METHODS: German national multicenter study of individuals with confirmed LCHAD/MTP deficiency identified by NBS between 1999 and 2020 or selective metabolic screening. Analyses focused on NBS results, confirmatory diagnostics, and long-term clinical outcomes. RESULTS: Sixty-seven individuals with LCHAD/MTP deficiency were included in the study, thereof 54 identified by NBS. All screened individuals with LCHAD deficiency survived, but four with MTP deficiency (14.8%) died during the study period. Despite NBS and early treatment neonatal decompensations (28%), symptomatic disease course (94%), later metabolic decompensations (80%), cardiomyopathy (28%), myopathy (82%), hepatopathy (32%), retinopathy (17%), and/or neuropathy (22%) occurred. Hospitalization rates were high (up to a mean of 2.4 times/year). Disease courses in screened individuals with LCHAD and MTP deficiency were similar except for neuropathy, occurring earlier in individuals with MTP deficiency (median 3.9 vs. 11.4 years; p = 0.0447). Achievement of dietary goals decreased with age, from 75% in the first year of life to 12% at age 10, and consensus group recommendations on dietary management were often not achieved. INTERPRETATION: While NBS and early treatment result in improved (neonatal) survival, they cannot reliably prevent long-term morbidity in screened individuals with LCHAD/MTP deficiency, highlighting the urgent need of better therapeutic strategies and the development of disease course-altering treatment.