Portuguese Neonatal Screening Program: A Cohort Study of 18 Years Using MS/MS.

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.

Portuguese Neonatal Screening Programme: A Retrospective Cohort Study of 18 Years of MS/MS.

INTRODUCTION: The Portuguese Neonatal Screening Programme (PNSP) identifies patients with rare diseases through nationwide screening. Currently, 27 diseases are diagnosed, amongst which are 24 Inborn Errors of Metabolism (IEM), covering approximately 100% of neonates (1). In 2004, the national laboratory implemented a new screening method, tandem mass spectrometry (MS/MS) to test for amino acids and acylcarnitines. This new protocol revolutionized the PNSP and allowed for the analysis of an increased number of IEM, with clear improvements in treatment timings and clinical outcomes (2). METHODS: From 2004 to 2022, 1 764 830 neonates were screened with MS/MS technology. Those who displayed biochemical profiles indicating an IEM were subjected to molecular characterization via genomic DNA extraction, PCR amplification, and direct Sanger sequencing method of dried blood spot samples. RESULTS/CASE REPORT: A cohort of 681 newborns were diagnosed with an IEM. MCAD deficiency is the most frequent, with 233 confirmed diagnoses, showing predominantly c.985A>G (p.K329E) mutation of the ACADM gene in homozygosity. Approximately 1/3 of the 33 confirmed cases of Glutaric Aciduria type I present homozygous for the c.1204C>T (p.Arg402Trp) mutation in GCDH. Around 60% of cases of MAT II/III deficiency display the dominant mutation of the MAT1A gene, c.791G>A (p.Arg264His). These genetic profiles and others were determined as diagnostic confirmation for 24 of the IEM screened. CONCLUSION: This data shows the molecular epidemiology of patients with confirmed IEM diagnosis identified by neonatal screening. Some diseases out of the scope of the PNSP were also detected as a differential diagnosis after biochemical suspicion in the dried blood spot sample. The retrospective analysis of the PNSP allows for an overview of 18 years of achievements accomplished by the national screening for IEM since MS/MS was implemented. For some pathologies with low incidence, it's difficult to trace a discernible pattern. However, presenting de novo mutations for these diseases might provide insights on how to approach different phenotypes. The aim of this work is to establish the molecular epidemiology of metabolic diseases screened.

Pilot Study on Newborn Screening for Spinal Muscular Atrophy.

INTRODUCTION: Newborn screening (NBS) in Portugal is a significant public health measure to provide early detection for specific disorders so that early treatment is possible. Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder that causes degeneration of anterior horn cells in the human spinal cord and subsequent loss of motor neurons. Its incidence is estimated in 1.6000-11.800 live births. A pilot study on 100.000 newborns is being carried out at the neonatal screening laboratory with the aim of determining the specificity, sensitivity, and feasibility of the SMA screening at the NBS laboratory in Portugal. METHODS: The study presented here was based on data obtained from neonatal screening, involving the analysis of 25.000 newborns. SMA screening is performed by a qualitative detection of exon 7 of the SMN1 gene. The assay was performed using a commercially available real-time PCR, the Eonis SMN1, TREC, and KREC kit. RESULTS/CASE REPORT: The dried blood spots of a total of 25.000 newborns were tested; among these newborns, two were diagnosed as having SMA with survival motor neuron 1 (SMN1) deletion. These two SMA-positive samples were sent to a specialized clinical centre and a peripheral blood sample was sent to the reference laboratory for confirmation of the exon 7 deletion and determination of the SMN2 copy number. CONCLUSION: Early diagnosis and intervention are important for SMA treatment to be effective; the treatment should be started at the pre-symptomatic stage of SMA. Thus, newborn screening for SMA is strongly recommended. Currently, targeted therapies for SMA are available, and attempts are being made worldwide to include SMA screening in newborns.

Role of RNA in Molecular Diagnosis of MADD Patients.

The electron-transfer flavoprotein dehydrogenase gene (ETFDH) encodes the ETF-ubiquinone oxidoreductase (ETF-QO) and has been reported to be the major cause of multiple acyl-CoA dehydrogenase deficiency (MADD). In this study, we present the clinical and molecular diagnostic challenges, at the DNA and RNA levels, involved in establishing the genotype of four MADD patients with novel ETFDH variants: a missense variant, two deep intronic variants and a gross deletion. RNA sequencing allowed the identification of the second causative allele in all studied patients. Simultaneous DNA and RNA investigation can increase the number of MADD patients that can be confirmed following the suggestive data results of an expanded newborn screening program. In clinical practice, accurate identification of pathogenic mutations is fundamental, particularly with regard to diagnostic, prognostic, therapeutic and ethical issues. Our study highlights the importance of RNA studies for a definitive molecular diagnosis of MADD patients, expands the background of ETFDH mutations and will be important in providing an accurate genetic counseling and a prenatal diagnosis for the affected families.

[Portuguese Newborn Screening Program.] / Programa portugués de cribado neonatal.

The Portuguese Newborn Screening Program is a public health program that started in 1979, screening for PKU, being totally supported by public funds. It's a non-mandatory well implemented program that testes about 99.9% of Portuguese newborns. In the actual screening panel encompasses 26 disorders, including inborn errors of metabolism, congenital hypothyroidism and cystic fibrosis. Sample collection is advised to be made at 3rd day of life and treatment begins in average by the 10th day. Every testes are performed in one single national laboratory, that processes about 88,000 samples/year. In the 41 years of program existence, more than 3,800,000 newborns were screened and 2,130 affected newborns detected, reflecting the positive impact of the Program in the population. Future perspectives include the increase of program value in terms of public health by optimizing the screening of the disorders already screened and evaluation the possibility of adding others.

El Programa Portugués de Cribado Neonatal es un programa de Salud Pública a nivel nacional, que tuvo su inicio en 1979 con el cribado de la fenilcetonuria y es financiado totalmente por el Estado portugués. Se trata de un programa no obligatorio, con una tasa de cobertura del 99,8%, en el que hoy en día se criban veintiséis enfermedades, incluyendo metabolopatías, hipotiroidismo congénito y fibrosis quística. La toma de muestra se hace al 3er día de vida y el tratamiento de los neonatos afectos empieza en torno al 10º día. Todos los análisis están centralizados en un único laboratorio, que procesa aproximadamente 88.000 muestras al año. En los últimos cuarenta y un años se cribaron más de 3.800.000 neonatos y se detectaron 2.130 niños afectados, lo que es un indicador del impacto del programa en la población. Los desafíos futuros incluyen la búsqueda de nuevas estrategias para incrementar el valor del programa, donde se evalúen nuevas enfermedades a cribar y la optimización del cribado actual.

Phenylketonuria in Portugal: Genotype-phenotype correlations using molecular, biochemical, and haplotypic analyses.

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.

Newborn screening for sickle cell disease in Europe: recommendations from a Pan-European Consensus Conference.

Sickle Cell Disease (SCD) is an increasing global health problem and presents significant challenges to European health care systems. Newborn screening (NBS) for SCD enables early initiation of preventive measures and has contributed to a reduction in childhood mortality from SCD. Policies and methodologies for NBS vary in different countries, and this might have consequences for the quality of care and clinical outcomes for SCD across Europe. A two-day Pan-European consensus conference was held in Berlin in April 2017 in order to appraise the current status of NBS for SCD and to develop consensus-based statements on indications and methodology for NBS for SCD in Europe. More than 50 SCD experts from 13 European countries participated in the conference. This paper aims to summarise the discussions and present consensus recommendations which can be used to support the development of NBS programmes in European countries where they do not yet exist, and to review existing programmes.

Cystic Fibrosis Newborn Screening in Portugal: PAP Value in Populations with Stringent Rules for Genetic Studies.

Newborn screening (NBS) for cystic fibrosis (CF) has been shown to be advantageous for children with CF, and has thus been included in most NBS programs using various algorithms. With this study, we intend to establish the most appropriate algorithm for CF-NBS in the Portuguese population, to determine the incidence, and to contribute to elucidating the genetic epidemiology of CF in Portugal. This was a nationwide three-year pilot study including 255,000 newborns (NB) that were also screened for congenital hypothyroidism (CH) and 24 other metabolic disorders included in the Portuguese screening program. Most samples were collected in local health centers spread all over the country, between the 3rd and 6th days of life. The algorithm tested includes immunoreactive trypsinogen (IRT) determination, pancreatitis associated protein (PAP) as a second tier, and genetic study for cases referred to specialized clinical centers. Thirty-four CF cases were confirmed positive, thus indicating an incidence of 1:7500 NB. The p.F508del mutation was found in 79% of the alleles. According to the results presented here, CF-NBS is recommended to be included in the Portuguese NBS panel with a small adjustment regarding the PAP cut-off, which we expect to contribute to the improvement of the CF-NBS performance. According to our results, this algorithm is a valuable alternative for CF-NBS in populations with stringent rules for genetic studies.

3-Methylcrotonyl-CoA carboxylase deficiency: Mutational spectrum derived from comprehensive newborn screening.

The deficiency of 3-methycrotonyl-CoA carboxylase (3-MCC; EC 6.4.1.4) is an autosomal recessive organic aciduria that is included in the newborn screening programs of several countries. This study reports data mainly obtained from the Portuguese newborn screening program collected over a ten-year period. Analysis of the MCCC1 and MCCC2 genes yielded 26 previously unreported mutations and a variant of clinically unknown significance. These mutations are discussed in the context of their likely impact on the function of the 3-MCC enzyme, with a view to exploring whether a phenotype-genotype correlation might be discerned. Further, these mutations were analysed in the context of what is known of the MCCC1 and MCCC2 mutational spectra, information that will be useful in both clinical and laboratory practice.

Newborn Screening for Homocystinuria Revealed a High Frequency of MAT I/III Deficiency in Iberian Peninsula.

Homocystinuria due to cystathionine ß-synthase deficiency or "classical homocystinuria" is a rare autosomal recessive condition resulting in altered sulfur metabolism with elevated methionine and homocysteine in plasma and homocystine in urine. This condition is characterized by a high clinical heterogeneity, which contributes to late clinical diagnosis, usually only made after irreversible damage has occurred. Treatment is effective if started before clinical symptoms. The analysis of methionine levels by tandem mass spectrometry (MS/MS) allows the newborn screening for homocystinuria, but false-positive results can be frequently obtained and lead to the unwanted identification of methionine adenosyl transferase (MAT I/III) deficiency. This latter condition is biochemically characterized by isolated persistent hypermethioninemia, accompanied in some individuals with slightly elevated levels of homocysteine in plasma. A dominant form of MAT I/III deficiency, associated with mutation p.R264H, seems to be very frequent in the Iberian Peninsula and usually has a clinically benign course. Both these metabolic disorders are screened in Galicia and Portugal since the introduction of the MS/MS technology, in 2000 and 2004, respectively, resulting in the identification of three patients with classical homocystinuria and 44 patients with MAT I/III deficiency. All but one heterozygous parent of MAT I/III patients, identified with the p.R264H mutation, are healthy adults around the age of 30/40. The implementation of a second-tier test for homocysteine in dried blood spots would considerably reduce the number of MAT I/III-deficient patients identified and improve the specificity and positive predictive value for classical homocystinuria screening.

Four years of expanded newborn screening in Portugal with tandem mass spectrometry.

INTRODUCTION: The Portuguese Neonatal Screening Programme (PNSP) was started in 1979 for phenylketonuria (2,590,700 newborns screened; prevalence 1:11,031) and, shortly after, for congenital hypothyroidism (2,558,455 newborns screened; prevalence 1:3,174). In 2004, expanded neonatal screening was implemented in the National Laboratory. The programme is not mandatory and has 99.8% coverage of the country (including Madeira and the Azores islands). MATERIAL AND METHODS: In the past 4 years, 316,243 neonates were screened with the use of tandem mass spectrometry (MS/MS) to test for selected amino acids and acylcarnitines. RESULTS: During this time, 132 patients were identified with 24 different inherited metabolic diseases (classic forms and variants). To date, the global frequency for all disorders integrated into the PNSP is estimated to be 1:1,380, with 1:2,396 for metabolic disorders. A total of 379 tests (0.12%) were classified as having false positive results, yielding an overall specificity of 99.9%. Despite the low frequency of several disorders, the positive predictive value of the overall MS/MS screening was found to be 26%, reflecting high diagnostic specificity of the method. Diagnostic sensitivity of extended screening for the different groups of disorders was 100%. Eight cases of maternal disorders [three glutaric aciduria type I, one carnitine transporter defect, and four 3-methylcrotonyl coenzyme A (CoA) carboxylase deficiency] were also detected through newborn screening. CONCLUSIONS: Our data support the advantage of a centralised laboratory for screening an elevated number of samples and making decisions if relying on a clinical network able to provide fast treatment and a good outcome in the screened cases.

Outcome of three cases of untreated maternal glutaric aciduria type I.

We report, for the first time, the outcome of three children born to two women with untreated glutaric aciduria type I (GA I). Isolated hypocarnitinemia in neonatal screening in one baby allowed the identification of the disease in his mother, who was undiagnosed so far and had had a previous daughter. The other baby was born to an already diagnosed mother who was not treated; newborn screening in the child reflected the metabolic state of the mother. Biochemical abnormalities returned to normal within one week. At the age of 4 months, neuroimaging showed Sylvian enlargement in both infants and bilateral temporal arachnoid cysts in one. Physical and neurological developments were normal for the three patients at ages 2 and 5 years. We conclude that long-term follow up will determine the true impact of GA I in such children.

Mutations c.459+1G>A and p.P426L in the ARSA gene: prevalence in metachromatic leukodystrophy patients from European countries.

In this multicentre study, we examined the prevalence of two mutations in the arylsulfatase A (ARSA) gene, i.e., c.459+1G>A and p.P426L, in 384 unrelated European patients presenting with different types of metachromatic leukodystrophy (MLD). In total, c.459+1G>A was found 194 times among the 768 investigated ARSA alleles (25%), whereas p.P426L was identified 143 times (18.6%). Thus, these two mutations accounted for 43.8% of investigated MLD alleles. Mutation c.459+1G>A was most frequent in late-infantile MLD patients (40%), while p.P426L was most frequent in adults (42.5%), which is consistent with earlier observations, although p.P426L was also found in a few late-infantile patients (0.9%), and c.459+1G>A was present in some adults (9%). Mutation c.459+1G>A is more frequent in countries situated at the western edges of Europe, i.e., in Great Britain and Portugal, and also in Belgium, Switzerland, and Italy, which is visible as a strand ranging from North to South, and additionally in Czech and Slovak Republics. Mutation p.P426L is most prevalent in countries assembled in a cluster containing the Netherlands, Germany, and Austria. In other Central European countries, the frequency of both c.459+1G>A and p.P426L ranges from 8 to 37.5%. Our study has confirmed that c.459+1G>A and p.P426L are the most frequently found MLD-causing mutations in Europe. The data about their prevalence reflect the population variability in Europe.

Missense mutations as a cause of metachromatic leukodystrophy. Degradation of arylsulfatase A in the endoplasmic reticulum.

Metachromatic leukodystrophy is a lysosomal storage disorder caused by a deficiency of arylsulfatase A (ASA). Biosynthesis studies of ASA with various structure-sensitive monoclonal antibodies reveal that some epitopes of the enzyme form within the first minutes of biosynthesis whereas other epitopes form later, between 10 and 25 min. When we investigated 12 various ASAs, with amino acid substitutions according to the missense mutations found in metachromatic leukodystrophy patients, immunoprecipitation with monoclonal antibodies revealed folding deficits in all 12 mutant ASA enzymes. Eleven of the 12 mutants show partial expression of the early epitopes, but only six of these show, in addition, incomplete expression of late epitopes. In none of the mutant enzymes were the late forming epitopes found in the absence of early epitopes. Thus, data from the wild-type and mutant enzymes indicate that the enzyme folds in a sequential manner and that the folding of early forming epitopes is a prerequisite for maturation of the late epitopes. All mutant enzymes in which the amino acid substitution prevents the expression of the late forming epitopes are retained in the endoplasmic reticulum (ER). In contrast, all mutants in which a single late epitope is at least partially expressed can leave the ER. Thus, irrespective of the missense mutation, the expression of epitopes forming late in biosynthesis correlates with the ability of the enzyme to leave the ER. The degradation of ER-retained enzymes can be reduced by inhibitors of the proteasome and ER alpha1,2-mannosidase I, indicating that all enzymes are degraded via the proteasome. Inhibition of degradation did not lead to an enhanced delivery from the ER for any of the mutant enzymes.

Adult onset metachromatic leukodystrophy without electroclinical peripheral nervous system involvement: a new mutation in the ARSA gene.

BACKGROUND: Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by the deficiency of arylsulfatase A (ARSA). Clinically, the disease is heterogeneous with respect to the age of onset, affection of peripheral and central nervous systems, and progression. OBJECTIVES: To analyze mutations in the ARSA gene of a patient with adult-onset MLD with no signs of peripheral polyneuropathy and to emphasize the clinical, neuroradiologic, neuropathologic, and genetic features of the disease. DESIGN: Case study of a patient clinically presenting with rapidly progressive dementia and behavioral abnormalities. We report the findings of clinical evaluation and neurophysiologic and neuropathologic studies of peripheral nerves; we also performed DNA sequence analysis, transfections, metabolic labeling, and immunoprecipitation of mutant ARSA polypeptides. SETTING: Genetic research and clinical unit, university hospital. RESULTS: Genetic analysis revealed homozygosity for a novel mutation in exon 3 of ARSA (F219V). This substitution leads to a misfolded unstable enzyme with a specific activity less than 1% of normal. There were no clinical or neurophysiologic signs of peripheral nervous system dysfunction. Typical neuropathologic signs for MLD were absent from nerve biopsy specimens. CONCLUSIONS: This novel mutation is associated with progressive psychocognitive impairment without clinical or electrophysiologic signs and only minor morphologic signs of peripheral nerve affection. The F219V substitution causes reduction in enzyme activity to an extent unexpected for an adult patient with MLD.

Prevalence of lysosomal storage diseases in Portugal.

Lysosomal storage diseases (LSDs) are a group of inherited metabolic disorders individually considered as rare, and few data on its prevalence has been reported in the literature. The overall birth prevalence of the 29 different LSDs studied in the Portuguese population was calculated to be 25/100000 live births, twice the prevalence previously described in Australia and in The Netherlands. The comparison of the prevalence profile of the LSDs presenting a prevalence higher than 0.5/100000 in the Portuguese, Dutch and Australian populations showed, in the Portuguese, the existence of a higher prevalence of GM2 gangliosidoses (B variant), mucolipidoses (II and III), Niemman-Pick type C and metachromatic leukodystrophy (MLD), and a lower prevalence of Pompe and Fabry. The highest prevalence value for a single LSD is the one of GM2 gangliosidoses (B variant), corresponding to 3/100000, a value which is significantly higher than the prevalence of the most frequent LSD in Dutch, Pompe disease (2/100000) and Australians, Gaucher's disease (GD) (1.8/100000). It is worth noting that the highest prevalence of GM2 gangliosidoses found in the Portuguese is mainly due to the existence of a unique subtype, the rare juvenile B1 variant.

ARSA-PD associated alleles in the Portuguese population: frequency determination and haplotype analysis.

Arylsulfatase A pseudodeficiency (ARSA-PD) may be related to increased susceptibility to neuro-psychiatric disorders. An association of allele 2417G/3352A with schizophrenia was found in a group of Portuguese patients. In the Portuguese population, at least one PD associated alteration exists in 18.3% of the ARSA alleles. Allele 2417G/3352G was invariably associated with a conserved haplotype, while 2417G/3352A and the rare 2417A/3352G alleles appeared on different haplotypes.

Oligomerization capacity of two arylsulfatase A mutants: C300F and P425T.

Arylsulfatase A (ARSA) is a lysosomal enzyme implicated in most cases of metachromatic leukodystrophy (MLD). The quaternary structure of ARSA is pH-dependent: at neutral pH, ARSA is a homodimeric protein; at lysosomal (acidic) pH, ARSA is homo-octameric. This dimer-octamer transition seems to be of major importance for the stability of the enzyme in the lysosomal milieu. Sedimentation analysis was used to study the oligomerization capacity of C300F and P425T-substituted ARSA, two MLD-associated forms of the enzyme displaying reduced lysosomal half-lives. P425T-ARSA displays a modest reduction in its octamerization capacity. In contrast, the C300F mutation strongly interferes with the octamerization process of ARSA but not with its dimerization capacity. Interestingly, a major fraction of dimeric ARSA-C300F is composed of covalently linked ARSA molecules, through a thiol-cleavable bond that probably involves Cys414 residues from each monomer. Our data support the notion that the reduced lysosomal half-life of some mutated forms of ARSA is related to deficient octamerization.

Biochemical characterization of two (C300F, P425T) arylsulfatase a missense mutations.

Metachromatic leukodystrophy (OMIM 250100) is a lysosomal storage disease caused by the deficiency of arylsulfatase A (ARSA, EC 3.1.6.8). This disease affects mainly the nervous system, because patients cannot degrade 3-O-sulfo-galactosylceramide (sulfatide), a major myelin lipid. Here we describe the characterization of the biochemical effects of two arylsulfatase A missense mutations, P425T and C300F. Transfection experiments demonstrate the expression of residual ARSA enzyme activity for P425T, but not for C300F substituted ARSA. Relative specific activity determination showed that the P425T substituted enzyme has retained about 12% of specific enzyme activity, whereas the C300F substituted enzyme is reduced to less than 1%. Pulse-chase experiments reveal that both mutant proteins are unstable, with a half life of less than 6 hr. Increased secretion upon addition of NH(4)Cl indicates that the mutant proteins can pass the Golgi apparatus and thus are not degraded in the endoplasmic reticulum (ER), but in the lysosomes. This is supported by experiments, which demonstrate the presence of mannose-6-phosphate residues on the oligosaccharide side chains of the mutant proteins. Addition of the cysteine protease inhibitor leupeptin increases the amount of ARSA activity in cells expressing the P425T substituted enzyme, whereas no increase in activity was seen with C300F substituted ARSA.