Birth prevalence of Cobalamin (Cbl) C or D defects in Portugal is an estimated 1:85,000, one of the highest worldwide. We compared the genotype/phenotype of patients identified with CblC or CblD before and after the implementation of expanded newborn screening. Twenty-five Portuguese CblC/D patients, 14 symptomatic and 11 identified through screening, were diagnosed using gas chromatography or tandem mass spectrometry. Molecular characterization was performed through the study of MMACHC and MMADHC genes. The most common MMACHC mutation, c.271dupA, was present in 100% of MMACHC alleles of all CblC screened patients, in contrast with the 61% identified before expanded newborn screening. All studied cases (except one, who presented a CblD deficiency) presented a CblC defect. More CblC late-onset patients were diagnosed before the introduction of newborn screening than in the post newborn screening era, probably because some early onset patients died without a definitive diagnosis. The molecular data found in this cohort contribute to the improvement of screening and diagnosis of Cbl defects and would enable a confirmatory diagnosis of these patients, reducing the need for complex, costly, laborious, and time-consuming biochemical/enzymatic tests.

The Portuguese Neonatal Screening Program (PNSP) conducts nationwide screening for rare diseases, covering nearly 100% of neonates and screening for 28 disorders, including 24 inborn errors of metabolism (IEMs). The study’s purpose is to assess the epidemiology of the screened metabolic diseases and to evaluate the impact of second-tier testing (2TT) within the PNSP. From 2004 to 2022, 1,764,830 neonates underwent screening using tandem mass spectrometry (MS/MS) to analyze amino acids and acylcarnitines in dried blood spot samples. 2TT was applied when necessary. Neonates with profiles indicating an IEM were reported to a reference treatment center, and subsequent biochemical and molecular studies were conducted for diagnostic confirmation. Among the screened neonates, 677 patients of IEM were identified, yielding an estimated birth prevalence of 1:2607 neonates. The introduction of 2TT significantly reduced false positives for various disorders, and 59 maternal cases were also detected. This study underscores the transformative role of MS/MS in neonatal screening, emphasizing the positive impact of 2TT in enhancing sensitivity, specificity, and positive predictive value. Our data highlight the efficiency and robustness of neonatal screening for IEM in Portugal, contributing to early and life-changing diagnoses.

Background: The impairment of the hepatic enzyme phenylalanine hydroxylase (PAH) causes elevation of phenylalanine levels in blood and other body fluids resulting in the most common inborn error of amino acid metabolism (phenylketonuria). Persistently high levels of phenylalanine lead to irreversible damage to the nervous system. Therefore, early diagnosis of the affected individuals is important, as it can prevent clinical manifestations of the disease.Methods: In this report, the biochemical and genetic findings performed in 223 patients diagnosed through the Portuguese Neonatal Screening Program (PNSP) are presented.Results: Overall, the results show that a high overlap exists between different types of variants and phenylalanine levels. Molecular analyses reveal a wide mutational spectrum in our population with a total of 56 previously reported variants, most of them found in compound heterozygosity (74% of the patients). Intragenic polymorphic markers were used to assess the haplotypic structure of mutated chromosomes for the most frequent variants found in homozygosity in our population (p.Ile65Thr, p.Arg158Gln, p.Leu249Phe, p.Arg261Gln, p.Val388Met, and c.1066‐11G>A).Conclusion: Our data reveal high heterogeneity at the biochemical and molecular levels and are expected to provide a better understanding of the molecular basis of this disease and to provide clues to elucidate genotype–phenotype correlations.