Inborn errors of metabolism (IEM) are rare diseases; most have incidences lower than 1 : 10,000. Early detection of IEMs is crucial in making correct and timely clinical decisions that can prevent serious consequences of the diseases or even death. Contrary to many western European countries, most south-eastern European countries do not have an expanded newborn screening (NBS) program using tandem mass spectrometry and only screen for a few diseases using conventional methods like immunoassays and fluorimetry. The aim of the work was to prepare an optimal strategy for the organization of the expanded NBS for IEMs in Slovenia, based on selected criteria and scoring system that we designed for the purpose of this study. The criteria and the scoring system will be formulated and evaluated on the basis of the experimental results of our research and in this way we will determine the set of diseases that should be included in the expanded NBS program for the Slovenian population. We conducted a retrospective study where samples of dried blood spots on the filter paper of 10,048 subjects were analysed. In the initial NBS stage we identified each IEM with tandem mass spectrometry and confirmed it with confirmatory analyses. For confirmations we also used the next-generation sequencing method (NGS). This method allows the simultaneous analysis of all genes associated with IEMs and thus represents the method of choice for the confirmation of the diseases identified in the NBS. Due to unclear NBS results, 113 follow-ups were evaluated at an outpatient visit where also additional confirmatory analyses were performed. Confirmation tests included the analysis of dried blood spots on the filter paper, the analysis of organic acids in the urine, the analysis of amino acids in plasma, and in selected cases also the analysis of enzymatic activity in fibroblasts and acylcarnitine profiling in fibroblasts. On the basis of the results of 10,048 subjects analysis and comparisons with European countries, we determined the cut-off values for individual metabolites and the ratios between them, and developed a scoring system that allowed us to identify subjects with the highest risk of developing each of the selected IEMs. Based on this, we have proposed a set of IEMs that should be included in the expanded NBS program in Slovenia. With confirmatory tests four new patients with IEMs were detected, namely a patient with glutaric aciduria type 1, a patient with very long-chain-acyl-CoA-ehydrogenase deficiency and two patients with 3-methylcrotonyl-CoA-carboxylase deficiency. In all cases, the NGS approach identified causative genetic variants, such as those that have not yet been described in the literature. Previously undescribed genetic variants were the causative genetic variant c.205-7-8delCTinsGC in the ACADVL gene and the benign genetic variant c.149_151dupCCA (p.Thr50dup) in the MCCC1 gene. The estimated cumulative incidence of IEM in Slovenia, which was calculated from the patients detected in our study and from the number of clinically detected cases, is 1 : 2,762 and is comparable to other European countries. We can conclude that the study successfully determined the appropriate cut-off values for each measured metabolite and the ratios between them, and defined the set of IEMs
for the Slovenian NBS program. We are one of the few groups that used the NGS genetic analysis for confirmatory testing in combination with other established confirmation tests for IEMs in the development of the national NBS strategy. The research also represents an important scientific contribution to the field of IEMs, as we identified and described two new genetic variants and for the Slovenian population developed an innovative scoring system for the selection of subjects with the highest risk for VBP. We have also determined the incidences of IEMs in Slovenia, which is the first estimate of the incidences of IEMs in South-Eastern Europe.
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