The Multi-Omic Approach to Newborn Screening: Opportunities and Challenges.

Newborn screening programs have seen significant evolution since their initial implementation more than 60 years ago, with the primary goal of detecting treatable conditions within the earliest possible timeframe to ensure the optimal treatment and outcomes for the newborn. New technologies have driven the expansion of screening programs to cover additional conditions. In the current era, the breadth of screened conditions could be further expanded by integrating omic technologies such as untargeted metabolomics and genomics. Genomic screening could offer opportunities for lifelong care beyond the newborn period. For genomic newborn screening to be effective and ready for routine adoption, it must overcome barriers such as implementation cost, public acceptability, and scalability. Metabolomics approaches, on the other hand, can offer insight into disease phenotypes and could be used to identify known and novel biomarkers of disease. Given recent advances in metabolomic technologies, alongside advances in genomics including whole-genome sequencing, the combination of complementary multi-omic approaches may provide an exciting opportunity to leverage the best of both approaches and overcome their respective limitations. These techniques are described, along with the current outlook on multi-omic-based NBS research.

Quantitation of total and free teriflunomide (A77 1726) in human plasma by LC-MS/MS.

The clinical activity of leflunomide, a drug used in the treatment of rheumatoid arthritis, is due to its active metabolite, teriflunomide. In vitro studies indicate that at least 99% of teriflunomide is expected to be protein bound in human plasma in vivo, leaving<1% in the unbound or 'free' state for clinical activity. To examine details of the relationships between leflunomide dosing and patient response, it is necessary to have an assay that is sufficiently sensitive to measure the minor fraction of free teriflunomide in patient samples. Therefore, we aimed to develop and validate an LC-MS/MS method for the measurement of teriflunomide, and use it to determine the total and free teriflunomide concentration in patients with rheumatoid arthritis. Teriflunomide and its deuterated internal standard were extracted from human plasma and separated using a reversed phase method with a C18 column. Detection was conducted with an API 3000 LC-MS/MS System by monitoring selected ions in negative ion MRM. Optimal detection occurred at m/z 269.1/160.0 (teriflunomide) and m/z 273.1/164.0 (teriflunomide-D4). Over a linear range of 5-500 µg/L, the inter-batch precision ranged from 1.9 to 8.8% and accuracy from -8.4 to 8.0%. The intra- and inter-batch assay precision for quality control samples ranged from 2.1-5.4% and 5.7-7.1% respectively. The procedure was applied to assess total and free plasma concentrations of teriflunomide in patients with rheumatoid arthritis. Free teriflunomide was approximately 0.11% of total teriflunomide, and there was a significant correlation (r2=0.724) between free and total teriflunomide concentrations. A validated, accurate and sensitive method was developed and successfully applied for the measurement of total and free teriflunomide concentration in human plasma samples. This method has been shown to be reproducible and sensitive and can be applied to clinical samples.

Determination of urinary oligosaccharides by high-performance liquid chromatography/electrospray ionization-tandem mass spectrometry: Application to Hunter syndrome.

The reaction of heparan sulfate (HS) and dermatan sulfate (DS) oligosaccharides with 1-phenyl-3-methyl-5-pyrazolone (PMP) yields hydrophobic derivatives that are amenable to separation by reversed-phase high-performance liquid chromatography (RP-HPLC) and analysis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). We describe here the development of an RP-HPLC-ESI-MS/MS assay for the measurement of di- to pentasaccharides derived from HS and DS in the urine of mucopolysaccharidosis (MPS) type II patients, as PMP derivatives. HPLC separation was performed on a 3-mum Alltima C18-LL column (50 x 2.1mm) using a gradient elution of up to 25% acetonitrile over 17 min, and an API-4000 mass spectrometer equipped with a turbo-ion-spray source was used in the negative ion multiple reaction monitoring mode for PMP-oligosaccharide determination. Using this method, we found that the derivatization kinetics of the oligosaccharides was influenced by the type of residue present at the reducing end (i.e., N-acetylglucosamine, N-acetylgalactosamine, or uronic acid). The elevation of each of the measured oligosaccharides in MPS II urine enabled complete discrimination of a cohort of MPS II patient urines from unaffected controls. This assay is rapid and reproducible and may be useful for the diagnosis of MPS II, and also for monitoring of disease progression and efficacy of therapy.

Examination of intravenous and intra-CSF protein delivery for treatment of neurological disease.

Mucopolysaccharidosis type IIIA is a neurodegenerative lysosomal storage disorder characterized by progressive loss of learned skills, sleep disturbance and behavioural problems. Absent or greatly reduced activity of sulphamidase, a lysosomal protein, results in intracellular accumulation of heparan sulphate. Subsequent neuroinflammation and neurodegeneration typify this and many other lysosomal storage disorders. We propose that intra-cerebrospinal fluid protein delivery represents a potential therapeutic avenue for treatment of this and other neurodegenerative conditions; however, technical restraints restrict examination of its use prior to adulthood in mice. We have used a naturally-occurring Mucopolysaccharidosis type IIIA mouse model to determine the effectiveness of combining intravenous protein replacement (1 mg/kg) from birth to 6 weeks of age with intra-cerebrospinal fluid sulphamidase delivery (100 microg, fortnightly from 6 weeks) on behaviour, the level of heparan sulphate-oligosaccharide storage and other neuropathology. Mice receiving combination treatment exhibited similar clinical improvement and reduction in heparan sulphate storage to those only receiving intra-cerebrospinal fluid enzyme. Reductions in micro- and astrogliosis and delayed development of ubiquitin-positive lesions were seen in both groups. A third group of intravenous-only treated mice did not exhibit clinical or neuropathological improvements. Intra-cerebrospinal fluid injection of sulphamidase effectively, but dose-dependently, treats neurological pathology in Mucopolysaccharidosis type IIIA, even when treatment begins in mice with established disease.

11-Hydroxy-3,3-dimethyl-7,12-dioxo-3,4,6,6a,7,12,12a,12b-octahydrobenz[a]anthracen-1-yl acetate.

The Diels-Alder reaction between 5-hydroxy-1,4-naphthoquinone and 5,5-dimethyl-3-vinyl-1,2-cyclohexadienyl acetate by endo addition gives the title compound, C(22)H(22)O(5), in 68% yield. This racemic diastereoisomer has the opposite regiochemistry to ochromycinone analogues produced previously and may allow access to a new type of anticancer-active saquayamycin analogue.

Amphibian peptides that inhibit neuronal nitric oxide synthase. Isolation of lesuerin from the skin secretion of the Australian Stony Creek frog Litoria lesueuri.

Two neuropeptides have been isolated and identified from the secretions of the skin glands of the Stony Creek Frog Litoria lesueuri. The first of these, the known neuropeptide caerulein 1.1, is a common constituent of anuran skin secretions, and has the sequence pEQY(SO3)TGWMDF-NH2. This neuropeptide is smooth muscle active, an analgaesic more potent than morphine and is also thought to be a hormone. The second neuropeptide, a new peptide, has been named lesueurin and has the primary structure GLLDILKKVGKVA-NH2. Lesueurin shows no significant antibiotic or anticancer activity, but inhibits the formation of the ubiquitous chemical messenger nitric oxide from neuronal nitric oxide synthase (nNOS) at IC(50) (16.2 microm), and is the first amphibian peptide reported to show inhibition of nNOS. As a consequence of this activity, we have tested other peptides previously isolated from Australian amphibians for nNOS inhibition. There are three groups of peptides that inhibit nNOS (IC(50) at microm concentrations): these are (a) the citropin/aurein type peptides (of which lesueurin is a member), e.g. citropin 1.1 (GLFDVIKKVASVIGGL-NH(2)) (8.2 microm); (b) the frenatin type peptides, e.g. frenatin 3 (GLMSVLGHAVGNVLG GLFKPK-OH) (6.8 microm); and (c) the caerin 1 peptides, e.g. caerin 1.8 (GLFGVLGSIAKHLLPHVVPVIAEKL-NH(2)) (1.7 microm). From Lineweaver-Burk plots, the mechanism of inhibition is revealed as noncompetitive with respect to the nNOS substrate arginine. When the nNOS inhibition tests with the three peptides outlined above were carried out in the presence of increasing concentrations of Ca(2+) calmodulin, the inhibition dropped by approximately 50% in each case. In addition, these peptides also inhibit the activity of calcineurin, another enzyme that requires the presence of the regulatory protein Ca(2+) calmodulin. It is proposed that the amphibian peptides inhibit nNOS by interacting with Ca(2+)calmodulin, and as a consequence, blocks the attachment of this protein to the calmodulin domain of nNOS.