A newborn Screening Programme for Inborn errors of metabolism in Galicia: 22 years of evaluation and follow-up.

BACKGROUND: There is a notable lack of harmonisation in newborn screening (NBS) programmes worldwide. The Galician programme for early detection of inborn errors of metabolism (IEM) was one of the first NBS programmes in Europe to incorporate mass spectrometry (July 2000). This programme currently screens for 26 IEMs in dried blood and urine samples collected 24-72 h after birth. RESULTS: In its 22-year history, this programme has analysed samples from 440,723 neonates and identified 326 cases of IEM with a prevalence of 1:1351. The most prevalent IEMs were hyperphenylalaninaemia (n = 118), followed by medium chain acyl-CoA dehydrogenase deficiency (MCADD, n = 26), galactosaemia (n = 20), and cystinurias (n = 43). Sixty-one false positives and 18 conditions related to maternal pathologies were detected. Urine samples have been identified as a useful secondary sample to reduce the rate of false positives and identify new defects. There were 5 false negatives. The overall positive value was 84.23%. The fatality rate over a median of 12.1 years of follow-up was 2.76%. The intelligence quotient of patients was normal in 95.7% of cases, and school performance was largely optimal, with pedagogic special needs assistance required in < 10% of cases. Clinical onset of disease preceded diagnosis in 4% of cases. The age at which first NBS report is performed was reduced by 4 days since 2021. CONCLUSIONS: This study highlights the benefits of collecting urine samples, reduce NBS reporting time and expanding the number of IEMs included in NBS programmes.

A glimpse into past, present, and future DNA sequencing.

Current advances in DNA sequencing technologies are dropping down sequencing cost while increasing throughput at a pace never shown before. Past-decade great milestones, as the establishment of a reference human genome (amongst others) and large-scale human genetic variation study in the 1000 Genome project are, in conjunction with the use of these techniques, triggering advances in many areas of basic and applied science. These tools, stored in and combined with the vast amount of information present in biological online databases are, with the use of automated interpretation and analysis tools, allowing the fulfillment of increasingly ambitious studies in many areas and also are democratizing the access to information, interpretation and technologies, being the first opportunity for researchers to assess the influence of genetics in complex events as multifactorial diseases, evolutionary studies, metagenomics, transcriptomics, etc. In this review, we present the current state of the art of these technologies, focusing on second generation sequencing, from sample and library preparation to sequencing chemistries and bioinformatic software available for final data analysis and visualisation, with its possible applications. We also make an overview of first and third generation, due to its historical importance and for being the upcoming future tools for genetic analysis, respectively.

Quantitative Clinical Chemistry Proteomics (qCCP) using mass spectrometry: general characteristics and application.

Proteomics studies typically aim to exhaustively detect peptides/proteins in a given biological sample. Over the past decade, the number of publications using proteomics methodologies has exploded. This was made possible due to the availability of high-quality genomic data and many technological advances in the fields of microfluidics and mass spectrometry. Proteomics in biomedical research was initially used in 'functional' studies for the identification of proteins involved in pathophysiological processes, complexes and networks. Improved sensitivity of instrumentation facilitated the analysis of even more complex sample types, including human biological fluids. It is at that point the field of clinical proteomics was born, and its fundamental aim was the discovery and (ideally) validation of biomarkers for the diagnosis, prognosis, or therapeutic monitoring of disease. Eventually, it was recognized that the technologies used in clinical proteomics studies [particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS)] could represent an alternative to classical immunochemical assays. Prior to deploying MS in the measurement of peptides/proteins in the clinical laboratory, it seems likely that traditional proteomics workflows and data management systems will need to adapt to the clinical environment and meet in vitro diagnostic (IVD) regulatory constraints. This defines a new field, as reviewed in this article, that we have termed quantitative Clinical Chemistry Proteomics (qCCP).

Development of electrospray ionization tandem mass spectrometry methods for the study of a high number of urine markers of inborn errors of metabolism.

RATIONALE: Rapid and specific screening methods to detect abnormal metabolites in biological fluids are important for the diagnosis of many Inborn Errors of Metabolism (IEM). In Galicia (N.W. Spain), where newborn screening (NBS) has long used both blood and urine dried samples, an expanded NBS by tandem mass spectrometry (MS/MS) begun in July 2000 analyzing amino acids and acylcarnitines in blood. The purpose of this study is the development of methods to widen and to complement the present NBS with the study of the selected metabolites in urine. METHODS: We studied and optimized the fragmentation of a total of 96 marking compounds of IEM, as well as 34 isotopically labeled internal standards (IS). The isobaric interferences were resolved with the use of alternative fragmentation in 14 of the 28 groups found. The methods were validated for 68 compounds following the recommendations of the NCCLS. RESULTS: We have developed electrospray ionization (ESI)- MS/MS methods in positive and negative ionization modes to detect selected metabolites in urine. The study was performed by direct injection of amino acids and acylcarnitines in positive mode, and organic acids, acylglycines, purines and pyrimidines in negative mode. Run times were 2.5 and 2.6 min, respectively, allowing the daily analysis of a high number of samples. CONCLUSIONS: The validated methods were proved effective for the simultaneous study of a large number of metabolites which are commonly present in urine samples and are used for detecting IEM. The evaluation was done by searching diagnostic profiles with multiple markers to increase sensitivity and specificity (e.g., acylcarnitines plus amino acids) or with specific urine markers (cystine, homogentisic acid, sialic acid, N-acetylaspartic acid, etc.).

Evaluation and long-term follow-up of infants with inborn errors of metabolism identified in an expanded screening programme.

Newborn screening (NBS) by tandem mass spectrometry started in Galicia (Spain) in 2000. We analyse the results of screening and clinical follow-up of inborn errors of metabolism (IEM) detected during 10 years. Our programme basically includes the disorders recommended by the American College of Medical Genetics. Since 2002, blood and urine samples have been collected from every newborn on the 3rd day of life; before then, samples were collected between the 5th and 8th days. Newborns who show abnormal results are referred to the clinical unit for diagnosis and treatment. In these 10 years, NBS has led directly to the identification of 137 IEM cases (one per 2060 newborns, if 35 cases of benign hyperphenylalaninemia are excluded). In addition, 33 false positive results and 10 cases of transitory elevation of biomarkers were identified (making the positive predictive rate 76.11%), and 4 false negative results. The use of urine samples contributed significantly to IEM detection in 44% of cases. Clinical symptoms appeared before positive screening results in nine patients (6.6%), four of them screened between days 5 and 8. The death rate was 2.92%; of the survivors, 95.5% were asymptomatic after a mean observation period of 54 months, and only two had an intellectual/psychomotor development score less than 85. Compared to other studies, a high incidence of type I glutaric aciduria was detected, one in 35,027 newborns. This report highlights the benefits of urine sample collection during screening, and it is the first study on expanded newborn screening results in Spain.

Trace elements in dried blood spots as potential discriminating features for metabolic disorder diagnosis in newborns.

Trace elements in dried blood spots (DBSs) from newborns were determined by laser ablation coupled with inductively coupled plasma mass spectrometry, and data were subjected to chemometric evaluation in an attempt to classify healthy newborns and newborns suffering from metabolic disorders. Unsupervised [principal component analysis (PCA) and cluster analysis (CA)] and supervised [linear discriminant analysis (LDA) and soft independent modeling by class analogy (SIMCA)] pattern recognition techniques were used as classification techniques. PCA and CA have shown a clear tendency to form two groups (healthy newborns and newborns suffering from metabolic disorders). LDA and SIMCA have predicted that 90.5% and 83.9% of originally grouped healthy newborn cases were correctly classified by LDA and SIMCA, respectively. In addition, these percentages were 97.6% (LDA) and 80.6% (SIMCA) for DBSs from newborns suffering from metabolic disorders. However, SIMCA has only detected one misclassified DBS from the healthy group, and the lower percentage is attributed to four DBSs from the healthy newborn group and five DBSs from newborns with disorders that were found as belonging to both categories (healthy newborns and newborns with disorders) in the training set. LDA also gave a percentage of grouped maple syrup urine disease (MSUD) cases correctly classified of 100%, although the percentage fells to 66.7% when classifying phenylketonuria (PKU) cases. Finally, essential elements such as Fe, K, Rb, and Zn were found to be matched (correlated) with the concentration of amino acids such as phenylalanine, valine, and leucine, biomarkers linked with MSUD and PKU diseases.

Acylcarnitine profile in neonatal hypoxic-ischemic encephalopathy: The value of butyrylcarnitine as a prognostic marker.

Optimal prognostic markers evaluating early neuroprotective interventions in neonatal hypoxic-ischemic encephalopathy (HIE) are lacking. This study was designed to assess the prognostic value of acylcarnitines in neonatal HIE.An observational cohort study was conducted over 10 years in 67 HIE. Variables analyzed included sex, blood cord pH, Apgar score, hypothermia treatment (yes/no), neuron-specific enolase (NSE) levels, and clinical outcome (neurological examination, brain magnetic resonance imaging [MRI], and electroencephalogram) before discharge and at 6 months. Acylcarnitine profiles were analyzed by tandem-mass spectrometry on dried-blood spots collected on day 3 for newborn screening. A cohort of healthy newborns was used as control group.HIE patients had significantly increased C4, C5, C5:1, C6, C6-OH, C8 levels (all P < .01) and decreased long-chain acylcarnitine levels (P < .03). Hypothermia treatment was associated with a decrease in C4 levels (p = 0.005) and an increase in most long-chain acylcarnitine levels (P < .01). A significant association was found between C4 levels and NSE on day 1 of hypothermia treatment (P = .002) and abnormal brain magnetic resonance imaging (MRI) at discharge (P = .037). In the hypothermia group, C4 levels decreased in patients with favorable outcomes but remained high in those who progressed unfavorably.C4 appears to be a good prognostic marker in HIE, as blood levels correlated with NSE levels and abnormal MRI findings. Furthermore, hypothermia did not lead to decreased levels in patients with adverse outcomes.

Clinical mass spectrometry proteomics (cMSP) for medical laboratory: What does the future hold?

BACKGROUND: Mass spectrometry (MS) methods are being widely used these days in medical laboratories for quantifying many small molecular analytes as well as for microbiological purposes. METHODS: Little use has been made so far, however, of MS for analyzing peptides and proteins in clinical laboratory (an approach known as clinical MS proteomics (cMSP)). The explanation for this situation may be that cMSP assays are more complex to implement than conventional assays, require large investments in terms of equipment and training, and have not yet been sufficiently validated for clinical applications. In addition, the protein analysis assays currently used in medical laboratories mostly meet both laboratory and clinical requirements in terms of analytical performances, ease of use, and turn-around-time. RESULTS: With the spread of MS methods in laboratories, increasing interest seems to be focusing on the development of MS for quantifying new analytes. MALDI-TOF MS methods have already been replacing classical methods of bacterial classification in clinical laboratories, for example, and this can be said to be an important step in this direction. CONCLUSIONS: In this paper, the literature available on the topic of clinical MS proteomics is reviewed and the pre-analytical, analytical, and post-analytical challenges which will have to be met in connection with this approach are discussed.

Assessment of a targeted resequencing assay as a support tool in the diagnosis of lysosomal storage disorders.

BACKGROUND: With over 50 different disorders and a combined incidence of up to 1/3000 births, lysosomal storage diseases (LSDs) constitute a major public health problem and place an enormous burden on affected individuals and their families. Many factors make LSD diagnosis difficult, including phenotype and penetrance variability, shared signs and symptoms, and problems inherent to biochemical diagnosis. Developing a powerful diagnostic tool could mitigate the protracted diagnostic process for these families, lead to better outcomes for current and proposed therapies, and provide the basis for more appropriate genetic counseling. METHODS: We have designed a targeted resequencing assay for the simultaneous testing of 57 lysosomal genes, using in-solution capture as the enrichment method and two different sequencing platforms. A total of 84 patients with high to moderate-or low suspicion index for LSD were enrolled in different centers in Spain and Portugal, including 18 positive controls. RESULTS: We correctly diagnosed 18 positive blinded controls, provided genetic diagnosis to 25 potential LSD patients, and ended with 18 diagnostic odysseys. CONCLUSION: We report the assessment of a next-generation-sequencing-based approach as an accessory tool in the diagnosis of LSDs, a group of disorders which have overlapping clinical profiles and genetic heterogeneity. We have also identified and quantified the strengths and limitations of next generation sequencing (NGS) technology applied to diagnosis.

Neuropeptide Kyotorphin (Tyrosyl-Arginine) has Decreased Levels in the Cerebro-Spinal Fluid of Alzheimer's Disease Patients: Potential Diagnostic and Pharmacological Implications.

In Alzheimer's disease (AD), besides the characteristic deterioration of memory, studies also point to a higher pain tolerance in spite of sensibility preservation. A change in the normal tau protein phosphorylation is also characteristic of AD, which contributes to the pathogenesis of the disease and is useful in early diagnosis. Kyotorphin (KTP) is an endogenous analgesic dipeptide (Tyr-Arg) for which there is evidence of eventual neuroprotective and neuromodulatory properties. The objective of this work was to study the possible correlation between KTP and phosphorylated tau protein (p-tau) levels in cerebro-spinal fluid (CSF) samples of AD patients. CSF samples were collected from 25 AD patients and 13 age-matched controls (N), where p-tau and KTP levels were measured. We found a statistically significant difference between p-tau/KTP values in AD and N groups with an inverse correlation between p-tau and KTP values in AD samples. These results suggest that in the future KTP may be a candidate biomarker for neurodegeneration and may be a lead compound to be used pharmacologically for neuroprotection.

Glutaric aciduria type I: outcome of patients with early- versus late-diagnosis.

Patients with Glutaric aciduria type 1 (GA-1) can be identified by newborn screening using tandem mass spectrometry. The clinical evolution of screened patients seems to be more favourable compared with those diagnosed later, although long-term evolution is still doubtful. We have evaluated the outcome in nine GA-1 patients diagnosed in our region during 12 years. Six were detected by newborn screening and 3 clinically. The birth prevalence was 1:35,027. High blood C5DC concentration, in 8/9 patients, was found, whereas all patients exhibited high concentration of this metabolite in urine. Therefore, urine C5DC was a good marker for the detection of this disease. Eight different mutations in the GCDH gene were identified, four of them were novel (p.R88H, p.Y398C, p.R372K, p.D220N); being p.R227P the mostcommon. Macrocephaly with enlarged frontotemporal subarachnoid space was present in 4/6 patients diagnosed by newborn screening, all these patients required high energy intake, and in two cases, enteral feeding during the first year of life was needed. One child had an intercurrent episode of feeding refuse with hypoglycemia at two years of age. The mean follow-up time of screened patients was 56 months, and patients still remain asymptomatic. However, after a mean follow-up of 97 months treatment efficacy was poor in unscreened patients, two of them showing a severe spastic tetraparesis. Plasma levels of lysine, tryptophan and carnitine, were the most useful biomarkers for the follow-up. Our data support that, early diagnosis and treatment strategies are essential measures for the good clinical evolution of GA-1 patients.

Newborn screening for medium-chain acyl-CoA dehydrogenase deficiency: regional experience and high incidence of carnitine deficiency.

BACKGROUND: Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common inherited defect in the mitochondrial fatty acid oxidation pathway, resulting in significant morbidity and mortality in undiagnosed patients.Newborn screening (NBS) has considerably improved MCADD outcome, but the risk of complication remains in some patients. The aim of this study was to evaluate the relationship between genotype, biochemical parameters and clinical data at diagnosis and during follow-up, in order to optimize monitoring of these patients. METHODS: We carried out a multicenter study in southwest Europe, of MCADD patients detected by NBS. Evaluated NBS data included free carnitine (C0) and the acylcarnitines C8, C10, C10:1 together with C8/C2 and C8/C10 ratios, clinical presentation parameters and genotype, in 45 patients. Follow-up data included C0 levels, duration of carnitine supplementation and occurrence of metabolic crises. RESULTS: C8/C2 ratio and C8 were the most accurate biomarkers of MCADD in NBS. We found a high number of patients homozygous for the prevalent c.985A > G mutation (75%). Moreover, in these patients C8, C8/C10 and C8/C2 were higher than in patients with other genotypes, while median value of C0 was significantly lower (23 µmol/L vs 36 µmol/L).The average follow-up period was 43 months. To keep carnitine levels within the normal range, carnitine supplementation was required in 82% of patients, and for a longer period in patients homozygotes for the c.985A>G mutation than in patients with other genotypes (average 31 vs 18 months). Even with treatment, median C0 levels remained lower in homozygous patients than in those with other genotypes (14 µmol/L vs 22 µmol/L).Two patients died and another three suffered a metabolic crisis, all of whom were homozygous for the c.985 A>G mutation. CONCLUSIONS: Our data show a direct association between homozygosity for c.985A>G and lower carnitine values at diagnosis, and a higher dose of carnitine supplementation for maintenance within the normal range. This study contributes to a better understanding of the relationship between genotype and phenotype in newborn patients with MCADD detected through screening which could be useful in improving follow-up strategies and clinical outcome.

Selenium speciation in cow milk obtained after supplementation with different selenium forms to the cow feed using liquid chromatography coupled with hydride generation-atomic fluorescence spectrometry.

The purpose of this paper is to develop an easy and quick on-line selenium speciation method (LC-UV-HG-AFS) in cow milk obtained after different supplementation to cow feed. This study focuses on selenium speciation in cow milk after the use of different selenium species (organic selenium as selenised yeast and inorganic selenium as sodium selenite) in the supplementation of forages. Separation was carried out on a muBondapack C(18) column with the positively charged ion-pairing agent tetraethylammonium chloride in the mobile phase. The optimization of pre-reduction conditions was carried out; this step was done with UV irradiation and a heating block to improve the reduction of the different Se-compounds. Variables such as exposure time, hydrochloric acid concentration and temperature were studied. The detection limits for SeCyst(2), Se(IV), SeMet and Se(VI) were 0.4, 0.5, 0.9 and 1.0mugl(-1), respectively. The proposed method was applied to cow milk samples. The milk samples obtained after an organic supplementation of feeding as selenised yeast present three species of selenium, SeCyst2, Se(IV) and SeMet, while only SeCyst2 and Se(IV) are present in milk samples obtained after an inorganic supplementation of feeding.

Study of the bioavailability of selenium in cows' milk after a supplementation of cow feed with different forms of selenium.

The purpose of the work described in this paper was to develop an easy and quick in-vitro method for comparing the bioavailability of selenium in cows' milk after different cow feed. The study focuses on bioavailability differences resulting from the use of different selenium species (organic selenium as selenised yeast and sodium selenite) for supplementation of forage. A procedure for determination of selenium in cows' milk and dialysates, by hydride-generation atomic-fluorescence spectrometry (HG-AFS) after microwave-assisted acid digestion, was optimised. The results show it is possible to obtain cows' milk enriched with selenium at different concentration without altering the original composition of the milk. The bioavailability was statistically greater for cows' milk obtained after supplementation of forage with organic selenium at levels of 0.4 and 0.5 microg Se g(-1) than for that obtained after supplementation with inorganic and organic selenium at levels of 0.2 and 0.3 microg Se g(-1).

Determination of total selenium and selenium distribution in the milk phases in commercial cow's milk by HG-AAS.

A procedure has been developed for determining the selenium in cow's milk using hydride generation-atomic absorption spectrometry (HG-AAS) following microwave-assisted acid digestion. The selenium distributions in milk whey, fat and micellar casein phases were studied after separating the different phases by ultracentrifugation and determining the selenium in all of them. The detection limits obtained by HG-AAS for the whole milk, milk whey and micellar casein were 0.074, 0.065 and 0.075 microg l(-1), respectively. The accuracy for the whole milk was checked by using a Certified Reference Material CRM 8435 whole milk powder from NIST, and the analytical recoveries for the milk whey and casein micelles were 100.9 and 96.9%, respectively. A mass balance study of the determination of selenium in the different milk phases was carried out, obtaining values of 95.5-100.8%. The total content of selenium was determined in 37 milk samples from 15 different manufacturers, 19 whole milk samples and 18 skimmed milk samples. The selenium levels found were within the 8.5-21 microg l(-1) range. The selenium distributions in the different milk phases were studied in 14 whole milk samples, and the highest selenium levels were found in milk whey (47.2-73.6%), while the lowest level was found for the fat phase (4.8-16.2%). A strong correlation was found between the selenium levels in whole milk and the selenium levels in the milk components.