In 1963, Robert Guthrie’s pioneering work developing a bacterial inhibition assay to measure phenylalanine in dried blood spots, provided the means for whole-population screening to detect phenylketonuria in the USA. In the following decades, NBS became firmly established as a part of public health in developed countries. Technological advances allowed for the addition of new disorders into routine programmes and thereby resulted in a paradigm shift. Today, technological advances in immunological methods, tandem mass spectrometry, PCR techniques, DNA sequencing for mutational variant analysis, ultra-high performance liquid chromatography (UPLC), iso-electric focusing, and digital microfluidics are employed in the NBS laboratory to detect more than 60 disorders. In this review, we will provide the current state of methodological advances that have been introduced into NBS. Particularly, ‘second-tier’ methods have significantly improved both the specificity and sensitivity of testing. We will also present how proteomic and metabolomic techniques can potentially improve screening strategies to reduce the number of false-positive results and improve the prediction of pathogenicity. Additionally, we discuss the application of complex, multiparameter statistical procedures that use large datasets and statistical algorithms to improve the predictive outcomes of tests. Future developments, utilizing genomic techniques, are also likely to play an increasingly important role, possibly combined with artificial intelligence (AI)-driven software. We will consider the balance required to harness the potential of these new advances whilst maintaining the benefits and reducing the risks for harm associated with all screening.
Aim: Cytokine polymorphisms may alter the fetal inflammatory response, increasing susceptibility to cerebral palsy (CP). This study investigates associations between selected inflammatory mediator and cytokine gene polymorphisms (Toll‐like receptor‐4 (TLR‐4) Asp299Gly, interleukin‐6 G‐174C and interleukin‐4 C‐589T) and CP from 443 CP infants and 883 control infants. Results were correlated with viral nucleic acids in the same samples. Results: At all gestational ages (GA), TLR‐4 was associated with a decreased risk of developing CP (homozygous/heterozygous odds ratio (OR) 0.70, 95% confidence interval (CI) 0.50–0.98) and interleukin (IL)‐6 was associated with an increased risk of developing hemiplegia (OR 1.38, 95% CI 1.05–1.83). For infants born 32–36 weeks GA, there was a tenfold increase in the risk of quadriplegic CP with homozygous/heterozygous IL‐6 (OR 10.42, 95% CI 1.34–80.82). Viral exposure in combination with IL‐4 in preterm infants was associated with a fourfold increased risk of quadriplegia (homozygous/heterozygous OR 4.25, 95% CI 1.21–14.95). In very preterm infants, the absence of detectable viral exposure in combination with IL‐4 decreased the risk of developing CP (homozygous/heterozygous OR 0.31, 95% CI 0.13–0.76). Conclusion: Polymorphisms in TLR‐4 may be associated with a decreased risk of CP. Polymorphisms in IL‐6 or IL‐4 may act as susceptibility genes, in the presence of viral exposure, for the development of hemiplegic and quadriplegic CP. These associations require confirmation but they suggest a hypothesis for CP causation due to double jeopardy from neurotropic viral exposure and genetic susceptibility to infection.
Methylmalonic aciduria is a rare disorder of organic acid metabolism with limited therapeutic options, resulting in high morbidity and mortality. Positive results from combined liver/kidney transplantation suggest, however, that metabolic sink therapy may be efficacious. Gene therapy offers a more accessible approach for the treatment of methylmalonic aciduria than organ transplantation. Accordingly, we have evaluated a lentiviral vector–mediated gene transfer approach in an in vivo mouse model of methylmalonic aciduria. A mouse model of methylmalonic aciduria (Mut−/−MUTh2) was injected intravenously at 8 weeks of age with a lentiviral vector that expressed a codon-optimized human methylmalonyl coenzyme A mutase transgene, HIV-1SDmEF1αmurSigHutMCM. Untreated Mut−/−MUTh2 and normal mice were used as controls. HIV-1SDmEF1αmurSigHutMCM-treated mice achieved near-normal weight for age, and Western blot analysis demonstrated significant methylmalonyl coenzyme A enzyme expression in their livers. Normalization of liver methylmalonyl coenzyme A enzyme activity in the treated group was associated with a reduction in plasma and urine methylmalonic acid levels, and a reduction in the hepatic methylmalonic acid concentration. Administration of the HIV-1SDmEF1αmurSigHutMCM vector provided significant, although incomplete, biochemical correction of methylmalonic aciduria in a mouse model, suggesting that gene therapy is a potential treatment for this disorder.
Abstract Background: The development of therapies for lysosomal storage disorders has created a need for biochemical markers to monitor the efficacy of therapy and methods to quantify these markers in biologic samples. In Pompe disease, the concentration of a tetrasaccharide, consisting of four glucose residues, is reputedly increased in urine and plasma, but faster and more sensitive methods are required for the analysis of this, and other oligosaccharides, from biologic fluids. Methods: We optimized the derivatization of storage oligosaccharides with 1-phenyl-3-methyl-5-pyrazolone for the measurement, by electrospray ionization tandem mass spectrometry, of oligosaccharide concentrations in urine (n = 6), plasma (n = 11), and dried-blood spots (n = 17) from Pompe-affected individuals. Age-matched control samples of urine (n = 10), plasma (n = 28), and blood spots (n = 369) were also analyzed. Results: The mean tetrasaccharide concentration was increased in urine from infantile-onset (0.69–12 mmol/mol of creatinine) and adult-onset (0.22–3.0 mmol/mol of creatinine) Pompe individuals compared with age-matched controls. In plasma samples, an increased tetrasaccharide concentration was observed in some infantile patients (up to 22 μmol/L) compared with age-matched controls (mean, 2.2 μmol/L). The method developed was sensitive enough to determine oligosaccharide concentrations in a single 3-mm blood spot, but no differences were observed between blood spots from control and Pompe-affected individuals. Conclusions: Measurements of oligosaccharide concentrations in urine by this new method have potential application for the diagnosis and monitoring of patients with Pompe disease. Plasma analysis may have limited application for infantile patients, but analysis of blood spots does not discriminate between controls and affected individuals.
The prostanoids are a family of biologically active lipids derived from the 20-carbon essential fatty acids (LCPUFA) which are involved in all aspects of the immune response including the resolution of inflammation. ω3-fatty acids, EPA DPA and DHA are anti-inflammatory, whilst the ω6-fatty acid, Arachidonic acid (AA) and its metabolites: 13(S)-HETE, TXB2, PGF2α and 6-k-PGF1α are pro-inflammatory. Liquid Chromatography Tandem Mass Spectrometry (LC-MSMS) allows analyses of multiple prostanoids with high accuracy using 3 mm blood spots. This method has never been used in JIA and may find biomarkers which can help predict disease activity and treatment response.
Background: Immunoglobulin G4-related disease (IgG4-RD) is a systemic disorder which may involve the lungs, airways and pleura. Objective: To describe novel findings in pleural involvement by IgG4-RD. Methods: A case series of patients with histologically proven IgG4-RD treated for pleural effusion in our clinic. Results: We identified 4 male patients (aged 68-84) with pleural effusion caused by IgG4-RD. Effusions were lymphocytic exudates in all cases, with high protein concentrations (5.6-7.4 g/dL). All patients had hyperglobulinemia, elevated serum immunoglobulin G (IgG) levels and elevated levels of IgG subclasses 1 and 4 (IgG1 and IgG4). In two patients, levels of adenosine deaminase (ADA) were measured in the effusion and were elevated (309 and 108 IU/L). Tuberculosis was excluded in both cases by pleural biopsy. Thoracic lymphadenopathy was prominent in all patients. One patient developed radiological findings compatible with rounded atelectasis (Figure). In all cases, effusion responded to corticosteroids therapy. Conclusions: IgG4-RD may cause an ADA-positive, lymphocytic exudate with a high protein concentration, resembling tuberculous effusion. Thoracic lymphadenopathy, hyperglobulinemia, and an increased total IgG, IgG1, IgG4 may suggest the diagnosis. Previously undescribed, IgG4-RD pleural inflammation may result in rounded atelectasis. Figure: Rounded atelectasis in a patient with IgG4-RD
Objective. To evaluate the use of protein markers using immune-quantification assays and of metabolite markers using tandem mass spectrometry for the identification, at birth, of individuals who have a lysosomal storage disorder. Methods. A retrospective analysis was conducted of Guthrie cards that were collected from newborns in Denmark during the period 1982–1997. Patients whose lysosomal storage disorder (LSD; 47 representing 12 disorders) was diagnosed in Denmark during the period 1982–1997 were selected, and their Guthrie cards were retrieved from storage. Control cards (227) were retrieved from the same period. Additional control cards (273) were collected from the South Australian Screening Centre (Australia). Results. From 2 protein and 94 metabolite markers, 15 were selected and evaluated for their use in the identification of LSDs. Glycosphingolipid and oligosaccharide markers showed 100% sensitivity and specificity for the identification of Fabry disease, α-mannosidosis, mucopolysaccharidosis (MPS) IVA, MPS IIIA, Tay-Sachs disease, and I-cell disease. Lower sensitivities were observed for Gaucher disease and sialidosis. No useful markers were identified for Krabbe disease, MPS II, Pompe disease, and Sandhoff disease. The protein markers LAMP-1 and saposin C were not able to differentiate individuals who had an LSD from the control population. Conclusions. Newborn screening for selected LSDs is possible with current technology. However, additional development is required to provide a broad coverage of disorders in a single, viable program.
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