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Abstract INTRODUCTION:
The Quebec Neonatal Urine Screening Program (QNUSP) began in 1973 at the CHUS in Sherbrooke. For nearly 52 years, this unique preventive genetic medicine program aimed for the early detection of more than 20 treatable hereditary metabolic diseases, which are divided into two groups: 1) urea cycle disorders and organic acidurias; and 2) disorders of metabolism and amino acid transport. The objective of newborn screening is to provide early diagnosis associated with therapeutic management before the onset of severe and irreversible clinical manifestations, such as intellectual disability, hyperammonemia, or metabolic acidosis that can lead to death. This unique program required the voluntary cooperation of parents to collect their baby's urine at 21 days of age and to mail the sample to the screening laboratory. Over the years, the average annual participation rate was between 84% and 93% and more than 3.7 million urine samples collected on filter paper have been analyzed. Screening has always been done qualitatively using thin-layer chromatography (TLC) which is a simple, rapid, reproducible and inexpensive technique. However, some drawbacks may be encountered using this technique. Its qualitative nature is not compatible with the participation to quality assurance programs, interferences can complicate the result interpretation, and the sensitivity is not adequate for the screening of certain diseases for which population screening would be beneficial, such as Triple H Syndrome. The use of tandem mass spectrometry (MS/MS) as a replacement technique for TLC would offer many advantages: 1) absolute quantitation using calibration curves, 2) direct normalization of urinary concentration via creatinine, 3) the possibility of participating in external quality assurance programs, 4) flexibility for adding diseases to be screened for, 5) better selectivity and therefore less interferences (creams, medications, etc.) and 6) better sensitivity enabling Triple H Syndrome screening by analyzing urinary orotic acid and uracil. It should be noted that there is a founder effect in Quebec for Triple H Syndrome, which reinforces the importance of urine screening, since the relevant biomarkers are not detected in blood. In the last few years, as part of research activities, we have thus been undertaking the technological enhancement from TLC to MS/MS for the screening of disorders originally screened as part of the QNUSP, Triple H Syndrome, and creatine deficiency disorders. This technology is now ready to be applied as part of an evaluative research project aiming to assess the feasibility in the context of a large-scale population-based project.
METHODS:
A leaflet explaining the evaluative research project will be given to parents at the birthplace. Parents who decide to participate will collect urine samples from their newborn on filter paper at 10 days of age and send it by regular mail to the research laboratory for analysis. A 5-cm diameter dried urine spot (DUS) will be eluted using a 0.01M NH4OH solution for 10-minutes on a rotary shaker. Twenty-five microliters of the eluate will be added to a solution containing the internal standards in a 96-well plate to perform the screening test, which includes 23 markers of inborn errors of metabolism (IEM) (amino acids, organic acids, and creatinine) and their respective internal standards. The screening test is suitable for the analysis of 500 samples/day (2.85 min per sample) and was devised using an Acquity I-Class/Xevo TQ-S micro UHPLC-MS/MS system (Waters Corp.) equipped with a sample organizer and operated in the flow injection mode. The multiple reaction monitoring acquisition mode was used, and results were analyzed using the NeoLynx software integrated to MassLynx v4.2. Two UHPLC-MS/MS second-tier tests where potential interferences were separated by chromatography were also developed and/or validated to confirm abnormal profiles from the screening test: 1) Analysis of a profile of 45 molecules comprising mainly of amino acids (Kairos Amino Acid Kit, Waters); 2) Analysis of a profile of 16 molecules, mainly organic acids, but also other biomarkers such as uracil and orotic acid (Triple H Syndrome), guanidinoacetic acid and creatine (creatine deficiency disorders) and creatinine (for normalization of the urine concentration).
RESULTS:
The analysis of DUS from 8 227 healthy newborns was performed as part of preliminary tests aiming to estimate reference values for the screening test and to perform a first evaluation of the feasibility to use the screening test as part of a large-scale population-based project. DUS from babies diagnosed with various IEM were also analyzed retrospectively and showed abnormal results for targeted biomarkers compared to established reference values. The analysis of a DUS from a newborn diagnosed with Triple H Syndrome indicates that newborn screening might be feasible in DUS using orotic acid and uracil normalized to creatinine as biomarkers.
CONCLUSION:
A screening test and two second-tier tests were developed and validated as part of a population-based newborn screening evaluative research project targeting IEM previously included in the QNUSP and analyzed by TLC, Triple H Syndrome, and creatine deficiency disorders. The feasibility to screen these IEM using MS/MS seems promising and will be evaluated as part of a 3-year research project. The founder effect in Quebec and the unicity of our approach for urine screening would be an important opportunity to better evaluate the prevalence of Triple H Syndrome and other disorders detectable in urine only and the outcomes of timely monitoring, and treatment in affected patients.
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= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
