José M. Fraga

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.

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.

Speciation of iron in breast milk and infant formulas whey by size exclusion chromatography-high performance liquid chromatography and electrothermal atomic absorption spectrometry.

Speciation of iron in milk was carried out by high performance liquid chromatography (HPLC) and electrothermal atomic absorption spectrometry (ETAAS). Milk whey was obtained and low molecular weight protein separation was performed by size exclusion chromatography (SEC) with a TSK Gel SW glass guard (Waters) pre-column and a TSK-Gel G2000 glass (Toso Haas) column. After studying water as a possible mobile phase, this mobile phase was carefully selected in order to avoid alterations of the sample and to make subsequent iron determination in the protein fractions easier by ETAAS. The proposed method is sensitive (limit of detection [LOD] and LOQ 1.4 and 4.7 microg l(-1), respectively) and precise (relative standard deviation [RSD]<10%). Iron is principally found in the proteins of 3 and 76 kDa in breast milk, and it is irregularly distributed in infant formulas.

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.

Determination of Finishing Oils in Acrylic Fibres by Near-infrared Reflectance Spectrometry

The potential of near-infrared diffuse reflectance spectrometry for quality control analyses in the textile industry was explored with a view to the quantification of finishing oils in acrylic fibres by partial least-squares regression, using a rotary cuvette system for recording spectra. Calibration was performed with a set of samples that encompassed every source of variability (linear density of the fibres, colour, concentration of the finishing oil), and the wavelength region where absorption was mostly due to the oil was used to construct several models from which that leading to the minimum relative standard error for a sample test set was selected. The results provided by various mathematical treatments [second- derivative, standard normal variate (SNV)] used to minimize scattering resulting from the differential linear density of the samples revealed no significant differences between prediction errors (only in the number of partial least-squares components). The model was used to quantify levels of finishing oil in routinely manufactured samples for a period of 6 months, during which time two batches showed poor predictions due to a new component appearing in the product. Modification of the calibration model to account for this component substantially increased robustness and allowed the accurate quantification of all batches manufactured after the model has been developed.

Risk factors for developing mineral bone disease in phenylketonuric patients

There is a compromised bone mass in phenylketonuria patients compared with normal population, but the mechanisms responsible are still a matter of investigation. In addition, tetrahydrobiopterin therapy is a new option for a significant proportion of these patients and the prevalence of mineral bone disease (MBD) in these patients is unknown. We conducted a cross-sectional observational study including 43 phenylketonuric patients. Bone densitometry, nutritional assessment, physical activity questionnaire, biochemical parameters, and molecular study were performed in all patients. Patients were stratified by phenotype, age and type of treatment. The MBD prevalence in phenylketonuria was 14%. Osteopenic and osteoporotic (n=6 patients) had an average daily natural protein intake significantly lower than the remaining (n=37) patients with PKU (14.33 ± 8.95 g vs 21.25 ± 20.85 g). Besides, a lower body mass index was found. There were no statistical differences in physical activity level, calcium, phosphorus and fat intake, and in phenylalanine, vitamin D, paratohormone, docosahexaenoic and eicosapentaenoic acid blood levels. Mutational spectrum was found in up to 30 different PAH genotypes and no relationship was established among genotype and development of MBD. None of the twelve phenylketonuric patients treated with tetrahydrobiopterin (27.9%), for an average of 7.1 years, developed MBD. Natural protein intake and blood levels of eicosapentaenoic acid were significantly higher while calcium intake was lower in these patients. This study shows that the decrease in natural protein intake can play an important role in MBD development in phenylketonuric patients. Therapy with tetrahydrobiopterin allows a more relaxed protein diet, which is associated with better bone mass.

Iron and zinc in hydrolised fractions of human milk and infant formulas using an in vitro method

Abstract The development of an in vitro method to simulate new-born digestion and to study iron and zinc bioavailability from human milk and cows milk based infant formulas was carried out. Enzyme treatment was conducted in two stages involving (1) pepsin at pH 5.0 followed by (2) pancreatin at neutral pH, where the incubation times were kept short to mimic the fast transit in the infants gastrointestinal tract. Solubility of trace elements was used to express bioavailability, and so analytes were determined in the fractions obtained after centrifugation by flame atomic absorption spectrometry (FAAS) using a high performance nebulizer. The results were compared to those obtained by performing gastric digestion at pH 2.0 for an adult, using various incubators to treat the sample and centrifugation or ultracentrifugation to separate soluble fractions. No differences in iron bioavailability from breast milk and infant formulas at different pHs could be detected due to the variability of the infant formulas analysed. However, zinc bioavailability from breast milk samples was higher than those obtained from infant formulas at the new born gastric pH.

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

BackgroundWith 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.MethodsWe 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.ResultsWe correctly diagnosed 18 positive blinded controls, provided genetic diagnosis to 25 potential LSD patients, and ended with 18 diagnostic odysseys.ConclusionWe 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.

Clinical and metabolic findings in patients with methionine adenosyltransferase I/III deficiency detected by newborn screening.

Persistent hypermethioninemia due to mutations in the MAT1A gene is often found during newborn screening (NBS) for homocystinuria due to cystathionine beta-synthase deficiency, however, outcomes and optimal management for these patients are not well established. We carried out a multicenter study of MAT I/III-deficient patients detected by NBS in four of the Spanish regional NBS programs. Data evaluated during NBS and follow-up for 18 patients included methionine and total homocysteine levels, clinical presentation parameters, genotypes, and development quotients. The birth prevalence was 1:1:22,874. At detection 16 of the 18 patients exhibited elevations of plasma methionine above 60 μmol/L (mean 99.9 ± 38 μmol/L) and the mean value in confirmation tests was 301 μmol/L (91-899) μmol/L. All patients were asymptomatic. In four patients with more markedly elevated plasma methionines (>450 μmol/L) total homocysteine values were slightly elevated (about 20 μmol/L). The average follow-up period was 3 years 7 months (range: 2-123 months). Most patients (83%) were heterozygous for the autosomal dominant Arg264His mutation and, with one exception, presented relatively low circulating methionine concentrations (<400 μM). Additional mutations identified in patients with mean confirmatory plasma methionines above 400 μM were Arg199Cys, Leu355Arg, and a novel mutation, Thr288Ala. During continued follow-up, the patients have been asymptomatic, and, to date, no therapeutic interventions have been utilized. Therefore, the currently available evidence shows that hypermethioninemia due to heterozygous MAT1A mutations such as Arg264His is a mild condition for which no treatment is necessary.

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.).